KR20230079095A - Process technologies for manufacturing and downstream purification of biological products - Google Patents

Abstract

In particular, biological production and downstream purification processes are provided herein.

Description

Process technologies for manufacturing and downstream purification of biological products

related application

This application, 35 U.S.C. Pursuant to §119(e), U.S. Provisional Application No. 63/077,766, filed on September 14, 2020, U.S. Provisional Application No. 63/154,108, filed on February 26, 2021, and filed on February 26, 2021 Claims the benefit of priority to US Provisional Application No. 63/154,109, the entire contents of each of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

New processes and methods for the manufacture and downstream purification of biological products are provided.

In particular, processes and apparatus for purifying biological products are provided herein. In aspects, provided herein is a process for purifying a biological product, comprising receiving a heterogeneous mixture comprising the biological product through an input line, and filtering in a dynamic filtration module. and removing impurities from the heterogeneous mixture. Impurities are removed from the heterogeneous mixture by feeding the biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure, thereby producing a filtrate containing the biological product. .

The dynamic filtration module substantially communicates with a dynamic filtration device, a target area configured to receive a heterogeneous mixture from at least one output head, and a vacuum collection system positioned between a feed reel and a collection reel. A membrane support member having a flat contact surface. The dynamic filtration device further includes a filter membrane extending between the supply reel and the collection reel together with at least one support member having a substantially planar contact surface. Purifying the biological product further includes passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains the biological product; The first module includes an affinity-based purification device. The affinity-based purification device has at least one first inlet and at least one first outlet configured to allow fluid flow between the at least one first inlet and the at least one first outlet through a mechanical rotation system. The mechanical rotating system includes a container carousel containing at least one individual container containing a suspension of beads. As described herein, the process comprises a process having at least one inlet for receiving a flow of a fraction comprising biological products from at least one outlet of a first module and from at least one first outlet of a first module. 2 Including further delivery to the module. The second module includes at least one free-flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, the second inlet and It is configured to allow continuous fluid flow between the second outlets to recover biological products.

In embodiments, the affinity-based purification device further comprises a lid system and a collection vessel system in fluid communication with the at least one individual vessel. For example, the lid system has at least one lid with gasket, at least two buffer inlets, a filling inlet, a gas inlet, and a venting valve. Also, the lead system is movable along the z-axis. The container carousel of the affinity-based purification apparatus can be rotated in a plane transverse to the z-axis, and the collection container can be moved along the z-axis.

As described herein, a container carousel of an affinity-based purification method includes at least one location for binding a biological product, at least one location for washing to remove unbound products, and at least one location for eluting and collecting the biological product. at least one location, and at least one regeneration location enabling recycling of beads.

For example, the bead surface of an affinity-based purification is linked to a protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer configured to selectively bind the biological product. The initial concentration of beads (eg, in an individual container that is positioned to bind the biological product) is in a concentration range of about 0.01% to about 25% by weight. Alternatively, the initial concentration of beads may be from about 0.01% to about 20%, from about 0.01% to about 10%, from about 0.01% to about 5%, from about 1% to about 20%, or in the range of about 5% to about 10% by weight. For example, the diameter of the beads ranges from about 0.2 μm to about 200 μm. In other examples, the beads have a diameter of about 0.2 μm to about 100 μm, about 1 μm to about 200 μm, about 10 μm to about 200 μm, about 20 μm to about 200 μm, about 30 μm to about 200 μm, about 50 μm to about 200 μm, or about 150 μm to about is 200 μm. Alternatively, the diameter of the beads is between about 1 μm and about 100 μm, or between about 50 μm and about 100 μm.

In embodiments, the beads (eg, beads of affinity-based purification) remain mobile during processing to maintain increased surface area available for binding. For example, the beads are kept separate (circulated or dispersed) in solution during the process (eg, the beads are individual beads). Also, a mobile bead may mean that the beads do not aggregate together, eg, at least two or more beads do not aggregate or group together. Additionally, migratory beads may mean that the beads are dispersed in solution to form small agglomerates that move freely. In contrast, the beads used herein are not packed, but retain mobility and move freely in solution.

In embodiments, a free flow electrophoretic device includes electrode channels including an anode electrode channel and a cathode electrode channel in liquid contact with a main separation channel through a wall gap.

The free flow electrophoresis device includes at least one electrode channel de-bubbler comprising at least one gas permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a primary bubble-free separation channel, and at least one There is one liquid circuit breaker. In embodiments, at least one bubble eliminator of the free flow electrophoresis device is configured to continuously remove O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removal of electrolytic bubbles from the electrode channels is essential to enable continuous operation over substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a water-tight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. . For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar. Unlike current methods, the process described herein removes gas bubbles prior to entering the main separation channels.

In embodiments, a liquid circuit breaker of a free flow electrophoretic device includes a pressurized vessel configured to maintain a flow rate and generate a droplet that disconnects the circuit from a solution connected to a voltage.

In embodiments, the purification process maintains an approximately constant flow rate in the dynamic filtration module, the first module, and the second module. For example, the flow rate ranges from about 0.1 mL/min to about 50 mL/min, or from about 5 mL/min to about 10 mL/min.

In embodiments, the process of purifying the biological product is performed at a temperature ranging from about 4°C to about 37°C.

In a further embodiment, the process may include at least two dynamic filtration modules, where each dynamic filtration module has a filter membrane comprising the same or a different pore size (e.g., a heterogeneous mixture is first Contact with a large pore size filter membrane (e.g., 0.45 μm), followed by contact with a next smaller pore size filter membrane (e.g., 0.2 μm).

In embodiments, the process comprises at least two free flow electrophoresis modules configured to operate in isoelectric focusing mode, zone electrophoresis mode, isotachophoresis mode, or a combination thereof. include The process described herein further comprises at least two dynamic filtration modules, at least two affinity based purification modules, or at least two free flow electrophoresis modules operated in parallel.

In aspects, provided herein is a dynamic filtration device for removing impurities from a biological product in a heterogeneous mixture. The apparatus includes a filter membrane extending between a supply reel and a collection reel, the filter membrane having a target area configured to receive the heterogeneous mixture from at least one output head configured to distribute the heterogeneous mixture to the target area. . The device's membrane support structure has a substantially flat contact surface for structurally supporting a portion of the filter membrane located between the supply and collection reels to create a target area. Additionally, the dynamic filtration device has at least one support member having a substantially planar contact surface for stabilizing transport of the filter membrane across the membrane support structure. A dynamic filtration device has a system configured to control the transport rate of the filter membrane. The dynamic filtration device includes a vacuum system having at least one vacuum line in communication with the membrane support structure and configured to apply a negative pressure gauge pressure to the dynamic filter membrane, wherein the negative pressure enables collection of the biological product-laden filtrate. In another example, the dynamic filtration device includes a wash buffer line.

In embodiments, the dynamic filtration device includes polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE). ), hydrophilic PTFE, or any combination thereof. The pore size of the filter membrane is in the range of about 0.1 μm to about 1 μm. In another example, the pore size is about 0.1 μm to about 0.9 μm, about 0.1 μm to about 0.8 μm, about 0.1 μm to about 0.7 μm, about 0.1 μm to about 0.6 μm, about 0.1 μm to about 0.5 μm, about 0.1 μm μm to about 0.4 μm, about 0.1 μm to about 0.3 μm, or about 0.1 μm to about 0.2 μm. As described herein, when two or more dynamic filtration devices are used, they may include filter membranes of similar or different sizes.

The dynamic filtration devices described herein include a membrane support structure having a series of parallel slots, for example from about 1 to about 10 parallel slots. In a specific example, there are 5 parallel slots in the membrane support structure.

Dynamic filtration devices described herein include a membrane support structure having a substantially planar contact surface, wherein the contact surface is, for example, from about 0.01 to about 0.1, from about 0.01 to about 0.05, or from about 0.05 to about 0.1 and As such, it is a measure of its static coefficient of friction. In a specific example, the static friction coefficient is 0.04.

In embodiments, the vacuum system of the dynamic filtration module is configured to apply a negative gauge pressure that is, for example, in the range of about -0.05 bar to about -0.98 bar.

In aspects, provided herein is a free flow electrophoretic device for separating a mixture into two or more fractions, at least one of which comprises a biological product. A free flow electrophoresis device comprising: at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet; at least one fluid channel configured to create an electric field gradient created between the two parallel plates and perpendicular to the direction of fluid flow; electrode channels comprising an anode electrode channel and a cathode electrode channel, wherein the electrode channels are configured to be connected to the main separation channel by liquid contact through a wall gap located between the electrode channels and the main separation channel; at least one electrode channel bubble eliminator comprising at least one gas permeable and hydrophobic membrane or porous material configured to remove electrolytic bubbles near the point of creation by a vacuum system to create a primary bubble free separation channel; at least one liquid circuit breaker configured to disconnect the liquid connected to the voltage prior to interacting with the at least one sensor or detector; active cooling system; and at least one collection vessel.

The free flow electrophoresis device described herein provides an electrode channel having a bubble eliminator, wherein the upper portion of the electrode channels is sealed with at least one gas permeable and hydrophobic membrane in communication with a vacuum system for removing air bubbles, the electrode The channels are open at the bottom of the channels and are configured to allow liquid contact of the electrode solution with the main separation channel solution through the wall gap.

The free flow electrophoretic device includes at least one electrode channel bubble eliminator comprising at least one gas permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a primary bubble free separation channel, and at least one liquid circuit breaker. there is

In embodiments, the free flow electrophoresis device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar. Unlike current methods, the process described herein removes gas bubbles prior to entering the main separation channels.

In embodiments, the wall gap (eg, the space in which the electrode channels are open at the bottom of the channels and configured to allow liquid contact of the electrode solution with the main separation channel solution) is between about 0.01 mm and about 0.25 mm. For example, the wall gap is between about 0.01 mm and about 0.2 mm, between about 0.01 mm and about 0.015 mm, or between about 0.01 mm and about 0.01 mm.

In another embodiment, a liquid circuit breaker of a free flow electrophoretic device includes a pressurized vessel configured to maintain a flow rate and generate a droplet that disconnects the circuit from a solution connected to a voltage.

In embodiments, the free flow electrophoresis device further includes an in-line sensor. For example, the in-line sensor may include a flow sensor, a pH sensor, a conductivity sensor, or any combination thereof.

In embodiments, a free flow electrophoresis device described herein includes at least two electrophoresis devices connected in series to enable stepwise purification and operated in an isoelectric focusing mode, a bandelectrophoresis mode, an isokinetic mode, or a combination thereof. It may include two free flow electrophoresis devices.

Also provided herein is the use of a free flow electrophoretic device for purifying a biological product from a mixture. The present invention further provides the use of a dynamic filtration device for purifying biological products from heterogeneous mixtures.

In aspects, provided herein is a process for purifying a biological product. The process includes receiving, through an input line, a heterogeneous mixture comprising a biological product. In embodiments, the process comprises continuously receiving, through an input line, a heterogeneous mixture comprising a biological product. In embodiments, the biological product is a protein or fragment thereof (polypeptide), antibody or fragment thereof, cytokine, chemokine, growth factor, enzyme, oligonucleotide, virus, adenovirus, adeno-associated virus (AAV), or lentivirus. include

In embodiments, the process includes removing impurities (eg, large impurities such as cells, cell debris, and aggregates) from the heterogeneous mixture by dynamic filtration. In some embodiments, the dynamic filtration process can be a continuous process to remove large impurities from a heterogeneous mixture. The dynamic filtration process comprises at least one dynamic filtration wherein a heterogeneous mixture comprising biological products is continuously supplied under negative pressure from at least one output head in fluid communication with an input line to a dynamic filtration module to produce a filtrate comprising biological products. contains the module

In embodiments, the process includes passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. For example, separation of a solution into two or more fractions includes one fraction containing biological products, and small impurities (e.g., host cell proteins, undesirable proteins and peptides, undesirable antibodies, (including undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachables, media components, and/or naturally occurring organic molecules with which they are naturally associated); One other fraction may be included.

In embodiments, the first module includes an affinity-based, magnetic purification apparatus. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet through the loop conveyor system. In another example, the first module has at least one first inlet and at least one first outlet, with continuous fluid flow between the first inlet and the first outlet via a pick and place robotics system. is configured to allow

In embodiments, the process directs a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module. The second module includes a charge-based magnetic purification device or an isoelectric point-based fluid purification device (also referred to herein as a free flow electrophoresis device). In another embodiment, the process directs the biological product from at least one first outlet of the first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. and the second module includes a charge-based magnetic purification device, or an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device. For example, the second module includes a charge-based magnetic purification device having at least one second inlet and at least one second outlet, and a continuous flow between the second inlet and the second outlet via a loop conveyor system. It is configured to allow fluid flow. In some examples, the second module includes a charge-based magnetic purification device having at least one second inlet and at least one second outlet, and a pick-and-place robotic system between the second inlet and the second outlet. It is configured to allow constant fluid flow. In another example, the second module includes a free flow electrophoresis device having at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet. . In embodiments, the processes described herein thereby purify a biological product.

In embodiments, herein is a process diagram for purifying a biological product comprising continuously receiving, via an input line, a heterogeneous mixture comprising the biological product, and removing large impurities from the heterogeneous mixture by dynamic filtration. Provided. In some embodiments, the dynamic filtration process can be a continuous process to remove large impurities from a heterogeneous mixture. The dynamic filtration process includes a dynamic filtration module generating a filtrate comprising biological product by continuously supplying biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure.

In embodiments, the process comprises passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. For example, the first module includes an affinity-based purification device. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet via a mechanical rotation system. In another example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet through a staged linear system. It consists of

In embodiments, the process directs a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module. and the second module comprises an isoelectric point-based fluid purification device, also referred to herein as a charge-based purification device, or a free flow electrophoresis device. In another embodiment, the process directs the biological product from at least one first outlet of the first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. and the second module comprises an isoelectric point-based fluid purification device, also referred to herein as a charge-based purification device or a free flow electrophoresis device. For example, the second module comprises a charge-based purification device having at least one second inlet and at least one second outlet, with continuous fluid flow between the second inlet and the second outlet via a mechanical rotation system. is configured to allow In some examples, the second module has at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet through a staged linear system. In another example, the second module includes a free flow electrophoresis device having at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet. . In embodiments, the processes described herein thereby purify a biological product.

In embodiments, herein is a process diagram for purifying a biological product comprising continuously receiving, through an input line, a heterogeneous mixture comprising the biological product, and removing large impurities from the heterogeneous mixture by dynamic filtration. Provided. In some embodiments, the dynamic filtration process can be a continuous process to remove large impurities from a heterogeneous mixture. The dynamic filtration process includes a dynamic filtration module generating a filtrate comprising biological product by continuously supplying biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure.

In embodiments, the process includes passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In embodiments, the first module includes an affinity-based fluid purification device. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet.

In embodiments, the process directs a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module. and the second module comprises an isoelectric point-based fluid purification device, also referred to herein as a charge-based fluid purification device, or a free flow electrophoresis device. In embodiments, the process directs a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module. and the second module comprises an isoelectric point-based fluid purification device, also referred to herein as a charge-based fluid purification device, or a free flow electrophoresis device. For example, the second module comprises a charge-based fluid purification device having at least one second inlet and at least one second outlet, to permit continuous fluid flow between the second inlet and the second outlet. It consists of In another example, the second module comprises a free flow electrophoresis device having at least one second inlet and at least one second outlet and configured to allow continuous fluid flow between the second inlet and the second outlet. do. In embodiments, the processes described herein thereby purify a biological product.

In embodiments, the process includes passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In embodiments, the first module comprises an affinity based tangential flow filtration (TFF) purification device. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet.

In embodiments, the process directs a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module. and the second module comprises an isoelectric point-based fluid purification device, also referred to herein as a charge-based TFF purification device, or a free flow electrophoresis device. In another embodiment, the process directs the biological product from at least one first outlet of the first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. wherein the second module comprises a charge-based TFF purification device, or an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device. For example, the second module comprises a charge-based TFF purification device having at least one second inlet and at least one second outlet and configured to allow continuous fluid flow between the second inlet and the second outlet. do. In another example, the second module includes a free flow electrophoresis device having at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet. . In embodiments, the processes described herein thereby purify a biological product.

As described herein, processes for removing large impurities from heterogeneous mixtures include centrifugation, disk-stack centrifugation, depth filtration, static filtration, tangential flow filtration, or any combination thereof. I never do that. Alternatively, the processes described herein may, for example, without intending to be limiting, a heterogeneous mixture comprising a biological product via an input line derived from an input from which any large impurities have been removed by a centrifuge and depth filtration process. can accommodate

As described herein, processes for continuously removing large impurities from heterogeneous mixtures include centrifugation, disk-stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. does not include Alternatively, the processes described herein may be used, for example, without intending to be limiting, from a continuous disk-stack centrifuge and an input derived from any continuous large impurity-removed input by a depth filtration process or a hydrocyclone process. A heterogeneous mixture comprising biological products can be continuously received through the line.

In embodiments, the processes described herein include purifying a biological product (eg, a monoclonal antibody) produced in a bioreactor. In some embodiments, the processes described herein include purifying a biological product produced continuously in a bioreactor. For example, a bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, the output bleed line providing continuous fluid flow from the bioreactor to the dynamic filtration module. It serves as an input line allowing flow. For example, bioreactor types include fed-batch bioreactors, perfusion bioreactors, chemostat bioreactors, or multi-compartment bioreactors, but , but not limited thereto. For example, the flow from the bioreactor discharge line is always fed to a downstream purification system. Alternatively, the processes described herein include purifying a biological product (eg, mRNA) that is not produced in a bioreactor.

In embodiments, provided herein is a method of purifying a biological product comprising receiving, via an input line, a heterogeneous mixture comprising the biological product, at least one in fluid communication with the input line under negative pressure. feeding the biological product from the output head to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity-based magnetic purification device. wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick and place robotic system; ; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes a charge-based magnetic purifying device, the second module has at least one second inlet and at least one second outlet, and the second module is operated by a loop conveyor system or a pick-and-place robot system. configured to allow continuous fluid flow between the inlet and the second outlet; and thereby purifying the biological product.

In another embodiment, a method of purifying a biological product is provided. The method comprises receiving, via an input line, a heterogeneous mixture comprising a biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions including at least one fraction containing a biological product, wherein the first module is an affinity-based magnetic purification device wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick and place robotic system. -; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, 2 configured to allow continuous fluid flow between the inlet and the second outlet; and thereby purifying the biological product.

In embodiments, a method of purifying a biological product is included, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity based purification device. and wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a mechanical rotation system; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes a charge-based purification device, the second module has at least one second inlet and at least one second outlet, and a mechanical rotation system is used to move between the second inlet and the second outlet. configured to allow fluid flow; and thereby purifying the biological product.

In another embodiment, a method of purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product from at least one output head in fluid communication with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity based purification device. and wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a mechanical rotation system; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; In this case, the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, 2 configured to allow continuous fluid flow between the inlet and the second outlet; and thereby purifying the biological product.

In embodiments, a method of purifying a biological product is included, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity based purification device. and wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a staged linear system; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; In this case, the second module includes a charge-based purification device, the second module has at least one second inlet and at least one second outlet, and a distance between the second inlet and the second outlet is formed through a stepwise linear system. configured to allow fluid flow; and thereby purifying the biological product.

In another embodiment, a method of purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity based purification device. and wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a staged linear system; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, 2 configured to allow continuous fluid flow between the inlet and the second outlet; and thereby purifying the biological product.

In embodiments, a method of purifying a biological product is included, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module is an affinity-based fluid purification device wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes a charge-based fluid purification device, the second module has at least one second inlet and at least one second outlet, and controls the fluid flow between the second inlet and the second outlet. configured to allow -; and thereby purifying the biological product.

In another embodiment, a method of purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module is an affinity-based fluid purification device wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, 2 configured to allow continuous fluid flow between the inlet and the second outlet; and thereby purifying the biological product.

In embodiments, a method of purifying a biological product is included, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product from at least one output head in fluid communication with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity based TFF purification device. wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module comprises a charge-based TFF purification device, the second module has at least one second inlet and at least one second outlet, and the fluid flow between the second inlet and the second outlet configured to allow -; and thereby purifying the biological product.

In another embodiment, a method of purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying a biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to produce a filtrate comprising the biological product; passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the first module comprises an affinity based TFF purification device. wherein the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet; conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module; At this time, the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, 2 configured to allow continuous fluid flow between the inlet and the second outlet; and thereby purifying the biological product.

Advantages of the processes and methods described herein include the ability to remove large impurities (eg, cells, cell debris, and aggregates) without membrane fouling or clogging. For example, clarification of cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or clogging of the filter membrane, making such methods unusable for long periods of time. It renders it unsuitable as a means for the continuous removal of large impurities from heterogeneous mixtures containing biological products by means of a continuous process. In contrast, the dynamic filtration devices described herein enable continuous removal of large impurities from heterogeneous mixtures containing biological products without fouling the membranes, because the active target area of the filter membrane is constantly being renewed.

In addition, since the entire process of producing and purifying the biological product can be continuous and a flow rate in the range of about 0.1 mL / min to about 50 mL / min can be maintained throughout the process, the process equipment and overall process footprint ) can occupy a much smaller footprint than current standard processes without sacrificing product throughput or yield on a kilogram/year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require a minimum of 200,000 square feet. For example, the flow rate of a process for purifying a biological product ranges from about 1 mL/min to about 10 mL/min. In some examples, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL/min to about 50 mL/min. In another example, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor discharge line. In another example, a process is provided wherein the flow rate of the step of continuously delivering the filtrate to the first module ranges from about 0.1 mL/min to about 50 mL/min. In another example, a process is provided wherein the flow rate of the step of continuously transferring a fraction comprising biological products from the first outlet to the second module ranges from about 0.1 mL/min to about 50 mL/min.

An important advantage of the processes and methods using magnetic resin beads (eg, magnetic agarose) or conventional resin beads (eg, agarose) described herein is that such systems are sanitized, recycled, and/or or that it does not require a conventional stationary phase or packed resin column (eg for standard chromatography) for regeneration. For example, such a system provides recycling and/or regeneration of resin beads (eg, magnetic or non-magnetic resin beads) to create infinite surface area of resin beads during operation, resulting in continuous and cost effective provides a way

In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, resin beads used during the purification of a biological product as described herein are continuously recycled and regenerated, such that the flow from the previous step in either the dynamic filtration module or the purification module without stopping the flow from the bioreactor discharge line can accommodate In other words, because the modules described in the present invention pass through these steps sequentially, they do not need to be left idle for sterilization, regeneration, and/or recycling after execution. The method requires column switching of several packed columns to accommodate a continuous input flow and enable regeneration and/or recycling of columns that have reached full capacity, as current methods have limited column capacity limitations due to resin packing constraints. It differs from current continuous chromatography methods in that it requires

Another advantage of the method described herein includes that the resin beads do not pack in the stationary phase, but rather the resin beads move. This mobility of the beads increases the surface area available for binding or association as substantially more of the resin bead surface is exposed and free to bind, eg, more biological products can bind to the beads. In addition, resin beads in conventionally packed columns (e.g., where the mobility of the beads is poor and the surface area is reduced) are exposed to high pressure differentials to create flow through the column. This high pressure differential compromises the integrity of the beads and shortens column life. The mobile resin beads of the presently described invention are subjected to substantially lower pressures and therefore are much softer to the fragile beads, thereby extending their lifespan. Additionally, this mobility increases the likelihood that the bead will regenerate (eg fully regenerate) and return to its initial state. This also makes the methods described herein more cost-effective because, for example, the resin is used more efficiently.

As described herein, the resin beads of the claimed methods and apparatus are mobile throughout the process. Existing chromatographic purification methods require, for example, column packing to sufficiently pack the beads together to create a stationary phase and high density. For example, the beads are kept separate (circulated or dispersed) in solution during the process (eg, the beads are individual beads). Also, a mobile bead may mean that the beads do not aggregate together, eg, at least two or more beads do not aggregate or group together. Additionally, migratory beads may mean that the beads are dispersed in solution to form small agglomerates that move freely. In contrast, the beads used herein are not packed, but retain mobility and move freely in solution.

An important advantage of the processes and methods using free flow electrophoresis described herein is that the system exhibits a "no product loss" process, i.e., the separation is a physicochemical property of the target biological product in an aqueous solution. is that the product does not need to interact with a resin or other purification moiety, since it occurs through interaction with an electric field according to the Another advantage is observed in the resolution of this approach (e.g., the ability to purify products with a high degree of physicochemical similarity), as products of higher purity can be obtained compared to conventional ion exchange chromatography. . For example, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Higher purity biological products can be obtained. In addition, the methods and apparatus described herein can increase purity (of biological products) compared to existing purification and chromatography methods. For example, the term "increased" in reference to a level refers to any percent increase above a control level (eg, a level of purity obtained after purification using conventional methods). In various embodiments, the increased level is at least or about 1%, 2%, 3%, 4%, or 5% increase, at least or about 10% increase, at least or about 15% increase in purity, compared to existing purification methods. % increase, at least or about 20% increase, at least or about 25% increase, at least or about 30% increase, at least or about 35% increase, at least or about 40% increase, at least or about 45% increase, at least or about 50% increase, at least or about 55% increase, at least or about 60% increase, at least or about 65% increase, at least or about 70% increase, at least or about 75% increase, at least or about 80% increase, at least or about 85% increase , at least or about a 90% increase, at least or about a 95% increase. In other examples of the invention, the purity of the biological product resulting from the methods and apparatus described herein is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold as compared to the purity of the biological product using standard commercial or chromatographic techniques. , 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x increase do.

In addition, separation based on the intrinsic physiochemical properties of biological products (e.g., isoelectric point, surface charge, net charge, zeta potential, electrophoretic mobility, electrostatic interaction, etc.) , antibodies or fragments thereof, cytokines, chemokines, growth factors, enzymes, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAV), or lentiviruses. Extend the usefulness of the approach.

In addition, the modular approach provides flexibility in process design to accommodate a wide range of biological products.

In embodiments, in the process described herein, during purification by dynamic filtration, a filtrate comprising biological products is produced and fed to a vacuum collection vessel capable of collecting about 50 mL to about 100 L under negative pressure. For example, a vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 50 L.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to an active target area of the filter membrane. For example, the at least one output head is a tube or slot die.

In embodiments, the at least one dynamic filtration module is a coaxial output head, a separate monoaxial output head, a separate slot die output head, a slot die output head with multiple openings, or any combination thereof. It may further include at least one additional input line for supplying the washing buffer through.

In some embodiments, a dynamic filtration module may include, for example, without intending to be limiting, an active or passive edge guide, a tension regulator (eg, a dancer), a brake and a tension detector, or any of these It includes elements known to those skilled in the art, such as any combination of.

In implementations, the process herein includes at least one output head (in fluid communication with an input line to the dynamic filtration module) capable of xy rastering or rθ rastering. For example, at least one output head is capable of xy rastering. In some examples, at least one output head is capable of rθ rastering. In another example, at least one output head is movable along the z-axis. In another example, at least one output head is capable of xy rastering and is movable along the z axis.

In embodiments, a dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, at least one vacuum line, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll comprises a rolled filter membrane, wherein the filter membrane is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride ( PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

In embodiments, the pore size of the rolled filter membrane is dependent on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

In embodiments, the width of the filter membrane roll is between about 10 mm and about 600 mm. For example, the width of a filter membrane roll may vary depending on the size of the dynamic filtration system, the size of the at least one output head, or the membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel, i.e., the filter membrane starts with a prefabricated roll and continues to an initially empty collection roll to form a reel-to-reel system. to-reel system).

In embodiments, the membrane support structure of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, polytetrafluoroethylene (PTFE)) and an opening that is continuous with the vacuum line. include For example, the static coefficient of friction ranges from about 0.01 to about 0.1, from about 0.01 to about 0.05, or from about 0.05 to about 0.1. For example, the membrane support structure of the dynamic filtration module includes openings. For example, the opening may include a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

In embodiments, a temperature control mechanism is included in the membrane support structure of the dynamic filtration module. The temperature control mechanism maintains the temperature between 4°C and 37°C. For example, during antibody purification, a temperature control mechanism maintains a temperature between 15°C and 37°C. Exemplary temperature control mechanisms include single loop controllers, multiloop controllers, closed loop controllers, proportional-integral-derivative (PID) controllers, Peltier devices, resistors. a resistive heating element, and/or a thermal chuck with a circulating water/propylene glycol jacket.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static friction coefficient (eg, PTFE, perfluoroalkoxy alkane (PFA)). For example, the static coefficient of friction ranges from about 0.01 to about 0.1, from about 0.01 to about 0.05, or from about 0.05 to about 0.1. For example, the support rods or rollers may be stationary or rotated. In some examples, the support rod may further include a bearing, for example a sleeve bearing.

In embodiments, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process includes a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced. Alternatively, similar results can be achieved with a single dynamic filtration device having at least two rolled filter membranes fed by separate feed reels, resulting in a filter over a target area (eg, an active target area). A stacked set of membranes is created, wherein the heterogeneous mixture is first contacted with a larger pore size filter membrane (eg, 0.45 μm) and then a next smaller pore size filter membrane (eg, 0.2 μm). ) come into contact with

In embodiments, the process described herein comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the The first module includes an affinity-based magnetic purification device. For example, the affinity-based magnetic purification device further includes a suspension of magnetic resin beads. The surfaces of the magnetic resin beads are linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

For example, the magnetic resin beads of the affinity-based magnetic purification device have a diameter of about 0.2 microns to about 200 microns. In other examples, the beads have a diameter of about 0.2 μm to about 100 μm, about 1 μm to about 200 μm, about 10 μm to about 200 μm, about 20 μm to about 200 μm, about 30 μm to about 200 μm, about 50 μm to about 200 μm, or about 150 μm to about 150 μm. It is about 200 μm. Alternatively, the diameter of the beads is between about 1 μm and about 100 μm, or between about 50 μm and about 100 μm. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the overall flow rate of the process. For example, purification of a monoclonal antibody may include magnetic resin beads ranging in size from about 40 microns to about 90 microns. Also, the concentration of the magnetic resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of magnetic resin beads may be about 1% by weight. In some instances, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1% to about 10% by weight. In another example, the binding capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, the process described herein comprises continuously conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module, wherein the second module is magnetically based on charge. and a purification device (eg, a positive charge and/or negative charge based magnetic purification device), wherein the charge based magnetic purification device further includes magnetic resin beads. For example, the surfaces of the magnetic resin beads may have cationic functional groups derived from the binding of positively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the positively charged functional groups include amines, cationic polymers, net positively charged peptides, net positively charged proteins, or any combination thereof. Alternatively, the surface of the magnetic resin beads may have anionic functional groups derived from the incorporation of negatively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the negatively charged functional groups include carboxyls, anionic polymers, net negatively charged peptides, net negatively charged proteins, oligonucleotides, or any combination thereof. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

In embodiments, the magnetic resin beads of the charge-based magnetic purification device are between about 0.2 microns and about 200 microns in diameter. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the overall flow rate of the process. For example, purification of a monoclonal antibody may include magnetic resin beads ranging in size from about 40 microns to about 90 microns. Also, the concentration of the magnetic resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of magnetic resin beads may be about 1% by weight. In some instances, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1% to about 10% by weight. In another example, the charge or electrostatic association capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, as described herein, the first (affinity-based magnetic purification) and/or second (charge-based magnetic purification comprising a positive charge- and/or negative charge-based magnetic purification device) module(s) One or both of them may also include at least one external magnetic field. For example, at least one external magnetic field includes a permanent magnet or an electromagnet. The at least one external magnetic field includes a magnetic field strength between about 0.01 Tesla and about 1 Tesla (eg, up to 1 Tesla). Alternatively, at least one external magnetic field is shielded.

In embodiments, a loop conveyor system is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. There are at least two transport containers filled with magnetic resin beads. For example, at least one of the at least two transport containers is placed in or near an external magnetic field to attract magnetic resin beads.

In embodiments, a pick-and-place robotic system is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. There are at least two transport containers filled with constructed magnetic resin beads. For example, at least one of the at least two transport containers is placed in or near an external magnetic field to attract magnetic resin beads.

In embodiments, the first (affinity-based magnetic purification) and/or the second (charge-based magnetic purification comprising a positive charge and/or negative charge based magnetic purification device) module operates in a fed-batch or perfusion mode. It further includes at least one tangential flow filtration system. For example, tangential flow filtration systems can be used to concentrate and buffer exchange fractions comprising biological products.

In embodiments, the process described herein comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the The first module includes an affinity-based purification device. For example, the affinity based purification device further comprises a suspension of resin beads. The surface of the resin beads is linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer.

For example, the resin beads of the affinity-based purification device have a diameter of about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody may include resin beads about 90 microns in size. In addition, the concentration of the resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of the resin beads may be about 1% by weight. In some instances, a purification of a monoclonal antibody may include resin beads having a concentration of about 1% to about 10% by weight. In another example, the binding capacity of resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof.

In another example, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, the process described herein comprises continuously conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module, wherein the second module is a charge-based purification device. (eg, a positive charge and/or negative charge based purification device), wherein the charge based purification device further includes resin beads. For example, the surfaces of the resin beads may have cationic functional groups derived from the binding of positively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the positively charged functional groups include amines, cationic polymers, net positively charged peptides, net positively charged proteins, or any combination thereof. Alternatively, the surface of the resin beads may have anionic functional groups derived from the incorporation of negatively charged functional groups to enable purification based on charge or electrostatic interactions. For example, negatively charged functional groups include carboxyls, anionic polymers, net negatively charged peptides, net negatively charged proteins, oligonucleotides, or any combination thereof.

In embodiments, the diameter of the resin beads of the charge based purification device is between about 0.2 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody may include resin beads about 90 microns in size. In addition, the concentration of the resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of the resin beads may be about 1% by weight. In some instances, a purification of a monoclonal antibody may include resin beads having a concentration of about 1% to about 10% by weight. In another example, the charge or electrostatic association capacity of resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, a mechanical rotating system (eg, a system that allows continuous fluid flow between a first and/or second inlet and a first and/or second outlet) includes a mixture comprising a biological product. comprising mobile resin beads configured to receive (e.g., continuously receive) and then transport the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof to a designated purification site. There are at least two containers (eg, filled with migratory resin beads).

In another embodiment, the system (eg, a staged linear system allowing for continuous fluid flow between the first and/or second inlets and the first and/or second outlets) includes a mixture comprising a biological product. comprising mobile resin beads configured to receive (e.g., continuously receive) and then process the resulting mixture comprising a biological product, resin beads, buffer, or any combination thereof (e.g., There are at least two containers (filled with migratory resin beads).

In embodiments, the first (affinity-based purification) and/or the second (charge-based purification comprising a positive charge and/or negative charge based purification device) module may be used to concentrate and buffer exchange a fraction comprising a biological product. and at least one tangential flow filtration system operating in either a feed or perfusion mode.

In embodiments, the process described herein comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein The first module is an affinity-based fluid purification device having at least one hybrid fluidic device or chip. In embodiments, at least one hybrid fluid element or chip has a cross-flow channel, at least one magnetic field, and at least one mechanical force generator. Additionally, the at least one mechanical force generator may include an ultrasonic transducer or piezoelectric component capable of generating a defined unidirectional force. In another example, the at least one external magnetic field includes a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, the at least one external magnetic field may exhibit a magnetic field strength of about 0.01 Tesla (T) to about 1 Tesla (eg, up to 1 Tesla). In another example, the magnetic field strength is about 0.01 T, about 0.1 T, or about 1 T. In another embodiment, at least one hybrid fluidic element or chip has a cross-flow channel, at least one magnetic field, and at least one dielectrophoretic electrode. At least one dielectrophoretic electrode is capable of inducing a defined unidirectional force. Additionally, the at least one external magnetic field includes a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, the at least one external magnetic field may exhibit a magnetic field strength between about 0.01 Tesla and about 1 Tesla (eg, up to about 1 Tesla).

In embodiments, the affinity-based fluid purification device further includes magnetic resin beads. The surface of the magnetic resin bead is linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

In embodiments, the diameter of the magnetic resin beads of the affinity-based fluid purification device is between about 0.2 microns and about 200 microns. For example, purification of a monoclonal antibody may include magnetic resin beads about 40 microns in size. Also, the concentration of the magnetic resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the initial concentration of magnetic resin beads may be about 1% by weight. In some instances, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1% to about 10% by weight. In another example, the binding capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, a process described herein comprises continuously conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module, wherein the second module is a charge-based fluid. It includes a purification device. For example, a charge-based fluid purification device has at least one hybrid fluid element or chip. The at least one hybrid fluid element or chip may have a cross-flow channel, at least one magnetic field, and at least one mechanical force generator. Additionally, the at least one mechanical force generator includes an ultrasonic transducer or piezoelectric component capable of generating a defined unidirectional force. In another example, the at least one external magnetic field includes a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, the at least one external magnetic field may exhibit a magnetic field strength of about 0.01 Tesla (T) to about 1 Tesla (eg, up to about 1 Tesla). In another example, the magnetic field strength is about 0.01 T, about 0.1 T, or about 1 T. In another embodiment, a hybrid fluidic device or chip may have a cross-flow channel, at least one magnetic field, and at least one dielectrophoretic electrode, wherein the at least one dielectrophoretic electrode is capable of inducing a defined unidirectional force. . Also, at least one external magnetic field includes a permanent magnet or an electromagnet. For example, the at least one external magnetic field includes a magnetic field strength between about 0.01 Tesla and about 1 Tesla (eg, up to about 1 Tesla).

In embodiments, the charge-based fluid purification device (eg, positive charge and/or negative charge based fluid purification device) further comprises a suspension of magnetic resin beads. The surface of the magnetic resin beads has cationic functional groups derived from the binding of positively charged functional groups to enable purification based on charge or electrostatic interactions. The positively charged functional groups include amines, cationic polymers, net positively charged peptides, net positively charged proteins, or any combination thereof. Alternatively, the magnetic resin bead surface may contain anionic functional groups derived from the incorporation of negatively charged functional groups to enable purification based on charge or electrostatic interactions. The negatively charged functional groups include carboxyls, anionic polymers, net negatively charged peptides, net negatively charged proteins, oligonucleotides, or any combination thereof. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

For example, the magnetic resin beads of the charge-based fluid purification device have a diameter of about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the flow rate of the process. For example, purification of a monoclonal antibody may include magnetic resin beads about 40 microns in size. Also, the concentration of the magnetic resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of magnetic resin beads may be about 1% by weight. In some instances, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1% to about 10% by weight. In another example, the charge or electrostatic association capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, the first (affinity-based fluid purification) module further includes at least one equilibration vessel enabling binding of the biological product to the magnetic resin bead surface, and from the magnetic resin bead surface. There is at least one low pH equilibration vessel enabling de-binding interactions of biological products.

In embodiments, the second (charge-based fluid purification comprising a positive charge and/or negative charge-based fluid purification device) module further includes a charge-based or electrostatic interaction of the biological product with the magnetic resin bead surface. There is at least one association equilibrium vessel enabling association, and at least one dissociation equilibrium vessel enabling dissociation of the biological product from the magnetic resin bead surface. For example, a number of dissociation equilibrium vessels are used in conjunction with a number of charge-based fluid purification devices to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

In embodiments, the magnetic resin beads described herein are recycled and reused. For example, the beads may be reused at least 2, 3, 4, or more times to purify a biological product. To enable recycling and reuse of the magnetic resin beads, at least one regeneration equilibration vessel may be used with a tangential flow filtration system to concentrate and buffer exchange the magnetic resin beads to return the magnetic resin beads to their pristine state.

As described herein, the first (affinity-based fluid purification) and/or the second (charge-based fluid purification comprising a positive charge and/or negative charge based fluid purification device) module is a hybrid for purifying a biological product. A microfluidic, mesofluidic, millifluidic, macrofluidic device or chip, or any combination thereof, for example, of at least one magnetic field and a piezoelectric component or dielectrophoretic electrode A hybrid microfluidic device comprising at least one.

In embodiments, the first (affinity based fluid purification) and/or the second (charge based fluid purification comprising a positive charge and/or negative charge based fluid purification device) module concentrates a fraction comprising a biological product. and at least one tangential flow filtration system operating in a fed-batch or per-through mode for buffer exchange.

In embodiments, the process described herein comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product, wherein the The first module includes an affinity-based TFF purification device. For example, an affinity-based TFF purification device has at least three tangential flow filtration systems in fluid communication.

In embodiments, the affinity-based TFF purification device further comprises a suspension of resin beads. The surface of the resin beads is linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer.

In embodiments, the diameter of the resin beads of the affinity-based TFF purification device is between about 10 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody may include resin beads about 90 microns in size. In addition, the concentration of the resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of the resin beads may be about 1% by weight to about 20% by weight. In another example, the binding capacity of the resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, the process described herein comprises continuously transferring a fraction comprising a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises charge-based TFF purification. devices (eg, positive charge and/or negative charge based TFF purification devices). For example, a charge-based TFF purification device has at least three tangential flow filtration systems in fluid communication.

In embodiments, the charge-based TFF purification device further comprises a suspension of resin beads. For example, the surfaces of the resin beads may have cationic functional groups derived from the binding of positively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the positively charged functional groups include amines, cationic polymers, net positively charged peptides, net positively charged proteins, or any combination thereof. Alternatively, the surface of the resin beads may have anionic functional groups derived from the incorporation of negatively charged functional groups to enable purification based on charge or electrostatic interactions. For example, negatively charged functional groups include carboxyls, anionic polymers, net negatively charged peptides, net negatively charged proteins, oligonucleotides, or any combination thereof.

In embodiments, the diameter of the resin beads of the charge-based TFF purification device is between about 0.2 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody may include resin beads about 90 microns in size. In addition, the concentration of the resin beads may be in the range of about 0.01% by weight to about 25% by weight. For example, the concentration of the resin beads may be about 1% by weight to about 20% by weight. In another example, the charge or electrostatic association capacity of resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, the first (affinity-based TFF purification) module further includes at least one equilibration vessel enabling binding of the biological product to the resin bead surface, and a debinding interaction of the biological product from the resin bead surface. There is at least one low pH equilibration vessel that allows for

In embodiments, the second (charge-based TFF purification comprising a positive charge and/or negative charge-based TFF purification device) module further enables association based on charge or electrostatic interaction of the biological product with the resin bead surface. and at least one dissociation equilibrium vessel enabling dissociation of the biological product from the resin bead surface. For example, multiple dissociation equilibrium vessels are used with multiple charge-based fluid purification devices to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

In embodiments, the resin beads described herein are recycled and reused. For example, the beads may be reused at least 2, 3, 4, or more times to purify a biological product. To enable recycling and reuse of the resin beads, at least one regeneration equilibration vessel may be used with a tangential flow filtration system to concentrate and buffer exchange the resin beads to return the resin beads to their pristine state.

In embodiments, the first (affinity-based TFF purification) and/or the second (charge-based TFF purification comprising a positive charge and/or negative charge based TFF purification device) module concentrates the fraction comprising the biological product and buffer exchanges it. It further includes at least one tangential flow filtration system for

In another embodiment, the process described herein comprises continuously conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module, wherein the second module is free herein. It includes an isoelectric point-based fluid purification device, also called a flow electrophoresis device. For example, a free flow electrophoresis device includes at least one fluid channel created between two parallel plates to operate in an isoelectric focusing mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. There is a fluid element. In another example, an isoelectric point-based fluid purification module includes a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a coarse pH gradient across the main separation channel. (eg, a pH range of about 2 to about 10); and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separating channel (e.g., from about 5 to about 8 pH range) and at least one second fluidic element. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6). Alternatively, the free flow electrophoresis device includes an electric field or electric field gradient perpendicular to the direction of fluid flow and a fluid channel created between two parallel plates to operate in a bandelectrophoresis or charge separation mode of operation, wherein the pH gradient There is at least one fluid element with no pH gradient.

In another example, an isoelectric point-based fluid purification module comprises a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7). at least one first fluid element; and at least one second fluid element comprising a fluid channel created between the two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (eg, less than pH 7). . In addition, the free flow electrophoresis device includes a fluid channel created between two parallel plates to operate in the isokinetic electrophoretic mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a spacer solution (e.g. There is at least one fluidic element comprising both an acidic pH gradient and a basic pH gradient separated by, for example, a NaCl solution.

In some implementations, the isoelectric point-based fluid purification module includes at least one first fluid element comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and two parallel plates and at least one second fluid element comprising a fluid channel created therebetween and an electric field or electric field gradient orthogonal to the direction of fluid flow, wherein each element is connected in series and is independent to enable purification. It can operate in operating mode. For example, at least one first free flow electrophoretic device can operate in isoelectric focusing mode and at least one second free flow electrophoretic device can operate in isokinetic mode, to increase separation resolution. They can be operated sequentially via a series connection.

In another embodiment, without intending to be limiting, an isoelectric based fluid purification module includes at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; A fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g., in the pH range of about 2 to about 10). at least one second fluid element; and a fluid channel created between the two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. and at least one third fluid element. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In another embodiment, the isoelectric point-based fluid purification device further comprises an active cooling system (e.g., a Peltier element, a thermal chuck with a circulating water/propylene glycol jacket) to control temperature and reduce Joule heat dissipation. makes it possible For example, the active cooling system can control cooling and/or Joule heat dissipation to enable operation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. For example, when isolating a biological product (eg, a monoclonal antibody), the temperature is maintained between about 4° C. and about 37° C.

In a further embodiment, the process of purifying the biological product may also include virus inactivation, virus filtration, tangential flow filtration (TFF), high performance tangential flow filtration (HP-TFF), ultrafiltration/diafiltration (UF/DF), filter sterilization, fill-finish, lyophilization, or any combination thereof, which is performed semi-continuously and downstream of the second module.

For example, the entire process described herein (for purifying a biological product) is carried out at a temperature within the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Additionally, commercial production scale processes for purifying biological products are performed in systems with footprints that occupy far fewer square feet than current technologies without sacrificing product throughput or yield on a kilograms per year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require at least 200,000 square feet.

The processes described herein are used to purify biological products, which include proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses, or lentiviruses, but are not limited thereto.

dynamic filtration module

In aspects, provided herein is a dynamic filtration module for removing large impurities from a biological product in a heterogeneous mixture. The dynamic filtration module continuously supplies biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure.

In embodiments, a dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, at least one vacuum line, a vacuum system, and at least one vacuum collection vessel.

The dynamic filtration module includes a rolled filter membrane extending between a supply reel and a collection reel, the filter membrane having a target area (eg, an active target area) configured to receive the heterogeneous mixture. For example, the filter membrane of the filter membrane roll is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), or a suitable material including, but not limited to, hydrophilic PTFE.

In embodiments, the pore size of the rolled filter membrane varies depending on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

In embodiments, the width of the filter membrane roll is between about 10 mm and about 600 mm. For example, the width of a filter membrane roll may vary depending on factors such as the size of the dynamic filtration system and the size of the membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel to create a reel-to-reel system. In operation, the heterogeneous mixture is applied to a new, unused target area of a filter membrane, also referred to herein as a "target area" (or "active target area"), wherein said filter membrane is a used filter membrane. The result of the collecting reel collecting the parts is their continuous movement across the membrane support structure at an appropriate transport speed. For example, feed reel movement is controlled by a servo motor coupled with a gear box that limits revolutions per minute (RPM) to a ratio of 200:1 to enable low membrane transfer speeds with high torque. do. Collecting reel movement is controlled by a servo motor coupled with a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. Additionally, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure consistent membrane transport rates and constantly changing diameters of the filter membrane rolls on both the feed and collect reels during operation.

For example, a thickness monitoring system or rotary encoder can be used to ensure consistent membrane transport rates. For example, the supply reel and the collection reel run in the same direction at the same speed. In another example, the supply and collection reels run in the same direction at different speeds. Other methods of transporting the filter membrane from the supply reel to the collection reel may be considered by those skilled in the coating and converting industry. In another example, two dynamic filtration systems are run in parallel. For example, two parallel dynamic filtration systems may allow continuous flow through the system while replacing used filter membrane rolls. Additionally, the two parallel dynamic filtration systems may equilibrate the entire vacuum collection vessel to atmospheric pressure to allow fluid flow from the bioreactor discharge line to the first purification module without interrupting the process of continuously receiving the heterogeneous mixture. there is.

Additionally, the dynamic filtration module includes a membrane support structure for supporting a target area (eg, an active target area) of the filter membrane when the filter membrane is subjected to negative pressure. The membrane support structure is located between the supply reel and the collection reel, has a mechanically flat contact surface derived from a material with a low static friction coefficient (eg PTFE), and has an opening continuous with the vacuum line. For example, the opening may include a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, PTFE, PFA). For example, the dynamic filtration module includes at least one support rod or roller with a mechanically flat contact surface to stabilize the movement of the filter membrane across the membrane support structure.

In embodiments, the membrane support structure of the dynamic filtration module includes a temperature control mechanism to maintain a desired temperature when there is evaporative cooling. The temperature control mechanism maintains a temperature between about 4°C and about 37°C. For example, during antibody purification, the temperature control mechanism maintains the temperature within the range of about 15°C to about 37°C.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to a target area (eg, an active target area) of the filter membrane. For example, the at least one output head is a tube or slot die.

In some embodiments, the dynamic filtration module includes at least one additional input line for supplying wash buffer through a coaxial output head, a separate monoaxial output head, a separate slot die output head, or a slot die output head with multiple openings. additionally includes

In some embodiments, the dynamic filtration module may include elements known in the coatings and converting industry, such as, but not intended to be limiting, active or passive edge guides, tension regulators (eg, dancers), brakes and tension detectors; or any combination thereof.

In embodiments, a dynamic filtration module includes a vacuum system in continuity with a membrane support structure to apply a negative pressure across a target area (eg, an active target area) of a filter membrane, wherein the negative pressure is applied to the membrane support structure. Allows a target area (eg, active target area) of the filter membrane to be crossed and allows collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. For example, two parallel dynamic filtration systems are run staggered to allow continuous flow through the system after the first vacuum collection vessel is fully filled and equilibrated with atmospheric pressure.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process includes a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced. Alternatively, a similar result can be achieved with a single dynamic filtration device having at least two rolled filter membranes fed by separate supply reels, resulting in a stacked set of filter membranes across the active target area; , where the heterogeneous mixture is first contacted with a larger pore size filter membrane (eg, 0.45 μm) and then with a smaller pore size filter membrane (eg, 0.2 μm).

Affinity-based magnetic purification module

In aspects, provided herein is an affinity-based magnetic purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, For example, it can be consistent and constant during steady state operation.

In embodiments, an affinity-based magnetic purification module comprises a suspension of magnetic resin beads, wherein a surface of the magnetic resin beads is configured, without limitation, to selectively bind to the biological product Protein A, Protein G , protein L, antigen protein, protein, receptor, antibody, or aptamer. For example, the magnetic resin beads are mobile.

Further, the affinity-based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. and a loop conveyor system including at least two transport containers filled with magnetic resin beads.

Alternatively, the affinity-based magnetic purification module continuously receives a mixture comprising a biological product and then transports the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. and a pick-and-place robotic system comprising at least two transport containers filled with magnetic resin beads configured to:

In embodiments, the affinity-based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable cleaning. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable elution of the biological product. Alternatively, at least one external magnetic field may be used to enable recycling of the magnetic resin beads. For example, mixing of the magnetic resin beads can be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

In embodiments, the affinity-based magnetic purification module includes at least one binding/washing buffer system.

In embodiments, the affinity-based magnetic purification module includes at least one elution buffer system.

In embodiments, the affinity-based magnetic purification module includes at least one magnetic resin bead regeneration buffer system.

In embodiments, the affinity-based magnetic purification module includes at least one aspirator system for removing waste solution from the at least two transport vessels.

In embodiments, the affinity-based magnetic purification module includes at least one sensor or detector.

In embodiments, the affinity-based magnetic purification module includes at least one fluid handling pump.

Positive charge-based magnetic purification module

In aspects, provided herein is a positive charge-based magnetic purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation.

In embodiments, a positive charge based magnetic purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. . For example, the magnetic resin beads are mobile.

Additionally, the positive charge-based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. and a loop conveyor system including at least two transport containers filled with resin beads.

Alternatively, the positive charge-based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. and a pick-and-place robotic system comprising at least two transport containers filled with constructed magnetic resin beads.

In embodiments, the positive charge based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable cleaning. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable dissociation and purification of the biological product. Alternatively, at least one external magnetic field may be used to enable recycling of the magnetic resin beads. For example, mixing of the magnetic resin beads can be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

In embodiments, the positive charge based magnetic purification module includes at least one association/wash buffer system.

In embodiments, the positive charge based magnetic purification module includes at least one dissociation buffer system. For example, several dissociation buffers differing in pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

In embodiments, the positive charge based magnetic purification module includes at least one magnetic resin bead regeneration buffer system.

In embodiments, the positive charge based magnetic purification module includes at least one aspirator system for removing waste liquid from at least two transport vessels.

In embodiments, a positive charge based magnetic purification module includes at least one sensor or detector.

In embodiments, the positive charge based magnetic purification module includes at least one fluid handling pump.

Negative charge based magnetic purification module

In aspects, provided herein is a negative charge based magnetic purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation.

In embodiments, a negative charge based magnetic purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises an anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength. . For example, the magnetic resin beads are mobile.

Further, the negative charge-based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. and a loop conveyor system including at least two transport containers filled with resin beads.

Alternatively, the negative charge-based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. and a pick-and-place robotic system comprising at least two transport containers filled with constructed magnetic resin beads.

In embodiments, the negative charge based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable cleaning. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable dissociation and purification of the biological product. Alternatively, at least one external magnetic field may be used to enable recycling of the magnetic resin beads. For example, mixing of the magnetic resin beads can be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

In embodiments, the negative charge based magnetic purification module includes at least one association/wash buffer system.

In embodiments, the negative charge based magnetic purification module includes at least one dissociation buffer system. For example, several dissociation buffers differing in pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

In embodiments, the negative charge based magnetic purification module includes at least one magnetic resin bead regeneration buffer system.

In embodiments, the negative charge based magnetic purification module includes at least one aspirator system for removing waste liquid from the at least two transport vessels.

In embodiments, the negative charge based magnetic purification module includes at least one sensor or detector.

In embodiments, the negative charge based magnetic purification module includes at least one fluid handling pump.

Affinity-based purification module

In aspects, provided herein is an affinity-based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, It can be consistent and constant during steady state operation.

In embodiments, an affinity-based purification module comprises a suspension of resin beads, wherein a surface of the resin beads is configured, without limitation, to selectively bind to the biological product: Protein A, Protein G, Protein L, It is linked to an antigenic protein, protein, receptor, antibody, or aptamer. For example, the resin beads are mobile.

In embodiments, an affinity-based purification module includes a lid system having at least one gasketed lid, wherein the at least one gasketed lid is capable of controlling a positive head pressure with a gas. It has at least one inlet for introducing. In addition, the lid system includes at least one vent port for equilibrating atmospheric pressure, at least one inlet for introducing a suspension of resin beads, and at least one for receiving a filtrate containing a biological product. There are at least two inlets for introducing an inlet, and/or a buffer system dispersing the resin beads to enable washing, elution therefrom, or regeneration of the resin beads. In some embodiments, at least one gasket lid also includes a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

In embodiments, an affinity-based purification module continuously receives a mixture comprising a mechanical rotational system, eg, a biological product, followed by the biological product, resin beads, buffer, or any combination thereof. and a carousel comprising at least two containers filled with resin beads configured to transport the resulting heterogeneous mixture. For example, a carousel is a rotating structure that holds and transports at least two containers to different process locations. In some examples, the mechanical rotation system is configured to mate with the lid system to enable pressurization and liquid handling. In another example, a mechanical rotation system controls movement or rotation in the xy plane.

In embodiments, each of the at least two vessels of the affinity-based purification module has a supported basement filter or filter membrane. For example, a bottom filter (or filter membrane) allows retention of resin beads during binding, debinding, washing, elution, and/or regeneration process steps. For example, at least two vessels may further include a valve to control liquid flow.

In another embodiment, the affinity-based purification module is a stepwise linear system, eg, continuously receiving a mixture comprising a biological product, followed by a biological product, resin beads, buffer, or any combination thereof. and at least two vessels filled with resin beads configured to process the resulting heterogeneous mixture. For example, at least two containers are configured to mate with a lid system to enable pressurization and liquid handling.

In embodiments, an affinity-based purification module is a collection system capable of interfacing with at least one of the at least two vessels of the mechanical rotation system to collect waste, a fraction comprising a biological product, or any combination thereof. includes For example, the collection system controls movement along the z-axis.

In another embodiment, the affinity-based purification module interfaces with at least one of the at least two vessels of the staged linear system to collect waste, a fraction comprising a biological product, or any combination thereof. includes For example, the collection system is connected to at least one of the at least two vessels.

In embodiments, an affinity-based purification module includes at least one gas. In some embodiments, without intending to be limited, the gas includes filtered nitrogen or compressed dry air. For example, the gas creates a pressure head of about 0.1 psi to about 30 psi.

In embodiments, the affinity-based purification module includes at least one binding/washing buffer system.

In embodiments, the affinity-based purification module includes at least one low pH elution buffer system.

In embodiments, the affinity-based purification module includes at least one resin bead regeneration buffer system.

In embodiments the affinity-based purification module includes at least one collection vessel.

In embodiments, an affinity-based purification module includes at least one sensor or detector.

In embodiments, an affinity-based purification module includes at least one fluid handling pump.

Positive Charge Based Purification Module

Also provided herein is a positive charge-based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product.

The positive charge based purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady. State can be consistent and constant during operation.

In embodiments, a positive charge based purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the resin beads are mobile.

In embodiments, a positive charge based purification module includes a lid system having at least one gasket lid, the at least one gasket lid comprising: at least one inlet for introducing a gas to control a positive head pressure; at least one inlet for introducing a suspension of resin beads; at least one vent port to equalize atmospheric pressure; at least one inlet for receiving a filtrate comprising biological products; and at least two inlets for introducing a buffer system dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. In some embodiments, the at least one gasket lid further includes a port for receiving an overhead agitation impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

In embodiments, the positive charge based purification module is a mechanical rotating system, eg, continuously receiving a mixture comprising a biological product, followed by production comprising the biological product, resin beads, buffer, or any combination thereof. and a carousel comprising at least two containers filled with resin beads configured to transport the heterogeneous mixture. For example, a carousel is a rotating structure that holds and transports at least two containers to different process locations. In some examples, the mechanical rotation system is configured to mate with a lid system to enable pressurization. In another example, a mechanical rotation system controls movement or rotation in the xy plane.

In embodiments, each of the at least two vessels of the positive charge based purification module has a supported bottom filter or filter membrane. For example, a base filter (or filter membrane) allows retention of resin beads during association, washing, dissociation, and/or regeneration process steps. For example, at least two vessels may further include a valve to control liquid flow.

In another embodiment, the positive charge based purification module is a stepwise linear system, eg, continuously receiving a mixture comprising a biological product, followed by production comprising the biological product, resin beads, buffer, or any combination thereof. and at least two vessels filled with resin beads configured to process the heterogeneous mixture. For example, at least two containers are configured to mate with a lid system to enable pressurization and liquid handling.

In embodiments, the positive charge based purification module comprises a collection system capable of interfacing with at least one of the at least two vessels of the mechanical rotating system to collect waste, a fraction comprising a biological product, or any combination thereof. include For example, the collection system controls movement along the z-axis.

In another embodiment, the positive charge-based purification module provides a collection system capable of interfacing with at least one of the at least two vessels of the staged linear system to collect waste, a fraction comprising a biological product, or any combination thereof. include For example, the collection system is connected to at least one of the at least two vessels.

In embodiments, an affinity-based purification module includes at least one gas. In some embodiments, without intending to be limited, the gas includes filtered nitrogen or compressed dry air. For example, the gas creates a pressure head of about 0.1 psi to about 30 psi.

In embodiments, the positive charge based purification module includes at least one association/wash buffer system.

In embodiments, the positive charge based purification module includes at least one dissociation buffer system. For example, several dissociation buffers differing in pH, ionic strength, or any combination thereof are used sequentially or sequentially to create a gradient dissociation effect.

In embodiments, the positive charge based purification module includes at least one resin bead regeneration buffer system.

In embodiments, the positive charge based purification module includes at least one collection vessel.

In embodiments, the positive charge based purification module includes at least one sensor or detector.

In embodiments, the positive charge based purification module includes at least one fluid handling pump.

Negative Charge Based Purification Module

In aspects, provided herein is a negative charge based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge based purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady. State can be consistent and constant during operation.

In embodiments, a negative charge based purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

In embodiments, a negative charge based purification module includes a lid system having at least one gasket lid, the at least one gasket lid comprising: at least one inlet for introducing a gas to control a positive head pressure; at least one vent port to equalize atmospheric pressure; at least one inlet for introducing a suspension of resin beads; at least one inlet for receiving a filtrate comprising biological products; and at least two inlets for introducing a buffer system dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. In some embodiments, the at least one gasket lid further includes a port for receiving an overhead agitation impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

In embodiments, a negative charge based purification module is a mechanical rotating system, eg, continuously receiving a mixture comprising a biological product, followed by production comprising the biological product, resin beads, buffer, or any combination thereof. and a carousel comprising at least two containers filled with resin beads configured to transport the heterogeneous mixture. For example, a carousel is a rotating structure that holds and transports at least two containers to different process locations. In some examples, the mechanical rotation system is configured to mate with a lid system to enable pressurization. In another example, a mechanical rotation system controls movement or rotation in the xy plane.

In embodiments, each of the at least two vessels of the positive charge based purification module has a supported bottom filter or filter membrane. For example, a base filter (or filter membrane) allows retention of resin beads during association, washing, dissociation, and/or regeneration process steps. For example, at least two vessels may further include a valve to control liquid flow.

In another embodiment, the positive charge based purification module is a stepwise linear system, eg, continuously receiving a mixture comprising a biological product, followed by production comprising the biological product, resin beads, buffer, or any combination thereof. and at least two vessels filled with resin beads configured to process the heterogeneous mixture. For example, at least two containers are configured to mate with a lid system to enable pressurization and liquid handling.

In embodiments, the positive charge based purification module comprises a collection system capable of interfacing with at least one of the at least two vessels of the mechanical rotating system to collect waste, a fraction comprising a biological product, or any combination thereof. include For example, the collection system controls movement along the z-axis.

In another embodiment, the positive charge-based purification module provides a collection system capable of interfacing with at least one of the at least two vessels of the staged linear system to collect waste, a fraction comprising a biological product, or any combination thereof. include For example, the collection system is connected to at least one of the at least two vessels.

In embodiments, an affinity-based purification module includes at least one gas. In some embodiments, without intending to be limited, the gas includes filtered nitrogen or compressed dry air. For example, the gas creates a pressure head of about 0.1 psi to about 30 psi.

In embodiments, the negative charge based purification module includes at least one association/wash buffer system.

In embodiments, the negative charge based purification module includes at least one dissociation buffer system. For example, several dissociation buffers differing in pH, ionic strength, or any combination thereof are used sequentially or sequentially to create a gradient dissociation effect.

In embodiments, the negative charge based purification module includes at least one resin bead regeneration buffer system.

In embodiments, the negative charge based purification module includes at least one collection vessel.

In embodiments, the negative charge based purification module includes at least one sensor or detector.

In embodiments, the negative charge based purification module includes at least one fluid handling pump.

Affinity-based fluid purification module

In aspects, provided herein is an affinity-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, For example, it can be consistent and constant during steady state operation.

In embodiments, an affinity-based fluid purification module comprises a suspension of magnetic resin beads, wherein a surface of the magnetic resin beads is configured, without limitation, to selectively bind to the biological product Protein A, Protein G , protein L, antigen protein, protein, receptor, antibody, or aptamer. For example, the magnetic resin beads are mobile.

In embodiments, an affinity-based fluid purification module comprises at least one equilibration vessel enabling binding of a biological product to a surface of a magnetic resin bead; and at least one of a cross-flow channel, at least one magnetic field, and a piezoelectric component or dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the biological product-bound magnetic resin beads from the heterogeneous mixture. one first hybrid cross-flow fluid element.

In embodiments, an affinity-based fluid purification module includes at least one low pH equilibration vessel allowing debinding of a biological product from a magnetic resin bead surface; and at least one of a cross-flow channel, at least one magnetic field, and a piezoelectric component or dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads from the unbound biological product and complete their elution. and at least one second hybrid cross-flow fluid element comprising:

In embodiments, the affinity-based fluid purification module further comprises at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange a fraction comprising a biological product.

In embodiments, an affinity-based fluid purification module includes at least two buffer systems.

In embodiments, an affinity-based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

In embodiments, an affinity-based fluid purification module includes at least one equilibration vessel configured to enable recycling of the magnetic resin beads.

In embodiments, an affinity-based fluid purification module includes at least one sensor or detector.

In embodiments, an affinity-based fluid purification module includes at least one fluid handling pump.

Positive Charge Based Fluid Purification Module

In aspects, provided herein is a positive charge based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation.

In embodiments, a positive charge based fluid purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. . For example, the magnetic resin beads are mobile.

In embodiments, the positive charge based fluid purification module includes at least one associative equilibrium vessel enabling association of a biological product with a surface of a magnetic resin bead; and at least one of a cross-flow channel, at least one magnetic field, and a piezoelectric component or dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads with which the biological product is associated from the heterogeneous mixture. one first hybrid cross-flow fluid element.

In embodiments, a positive charge based fluid purification module includes at least one dissociation equilibrium vessel allowing dissociation of a biological product from a magnetic resin bead surface; and at least one of a cross-flow channel, at least one magnetic field, and a piezoelectric component or dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads from the dissociated biological product and complete purification thereof. and at least one second hybrid cross-flow fluid element. For example, several dissociation equilibrium vessels containing separate buffers of different pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

In embodiments, the positive charge based fluid purification module further comprises at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange a fraction comprising biological products.

In embodiments, a positive charge based fluid purification module includes at least two buffer systems.

In embodiments, the positive charge based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

In embodiments, a positive charge based fluid purification module includes at least one equilibration vessel configured to enable recycling of the magnetic resin beads.

In embodiments, a positive charge based fluid purification module includes at least one sensor or detector.

In embodiments, a positive charge based fluid purification module includes at least one fluid handling pump.

Negative Charge Based Fluid Purification Module

In aspects, provided herein is a negative charge based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation.

In embodiments, a negative charge based fluid purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises an anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength. . For example, the magnetic resin beads are mobile.

In embodiments, a negative charge based fluid purification module comprises at least one associative equilibrium vessel enabling association of a biological product with a surface of a magnetic resin bead; and at least one of a cross-flow channel, at least one magnetic field, and a piezoelectric component or dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads with which the biological product is associated from the heterogeneous mixture. one first hybrid cross-flow fluid element.

In embodiments, a negative charge based fluid purification module comprises at least one dissociation equilibrium vessel allowing dissociation of a biological product from a magnetic resin bead surface; and at least one of a cross-flow channel, at least one magnetic field, and a piezoelectric component or dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads from the dissociated biological product and complete purification thereof. and at least one second hybrid cross-flow fluid element. For example, several dissociation equilibrium vessels containing separate buffers of different pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

In embodiments, the negative charge based fluid purification module further comprises at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange the fraction comprising the biological product.

In embodiments, a negative charge based fluid purification module includes at least two buffer systems.

In embodiments, the negative charge based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

In embodiments, a negative charge based fluid purification module includes at least one equilibration vessel configured to enable recycling of the magnetic resin beads.

In embodiments, a negative charge based fluid purification module includes at least one sensor or detector.

In embodiments, a negative charge based fluid purification module includes at least one fluid handling pump.

Affinity-based TFF purification module

In aspects, provided herein is an affinity-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is maintained during steady state operation. consistent and consistent

In embodiments, an affinity-based TFF purification module comprises a suspension of resin beads, wherein a surface of the resin beads is configured, without limitation, to selectively bind to the biological product Protein A, Protein G, Protein L , antigenic proteins, proteins, receptors, antibodies, or aptamers. For example, the magnetic resin beads are mobile.

In embodiments, an affinity-based TFF purification module comprises at least one equilibration vessel allowing binding of a biological product to a resin bead surface; and at least one first tangential flow filtration system for separating the biological product bound resin beads from the heterogeneous mixture.

In embodiments, an affinity-based TFF purification module includes at least one low pH equilibration vessel that enables debinding of a biological product from a resin bead surface; and at least one second tangential flow filtration system for separating the resin beads from the unbound biological product and completing their elution.

In embodiments, an affinity-based TFF purification module comprises at least one regeneration equilibration vessel; and at least one third tangential flow filtration system that allows concentration and buffer exchange of the resin beads to return the resin beads to their initial state to enable recycling and reuse of the resin beads.

In embodiments, an affinity-based TFF purification module comprises at least one collection vessel; and at least one fourth tangential flow filtration system for purifying the biological product by enabling concentration and buffer exchange of the biological product.

In embodiments, the at least one equilibration vessel, the at least one low pH equilibration vessel, and the at least one regeneration equilibration vessel of an affinity-based TFF purification module provide continuous flow of filtrate through at least one additional vessel in a parallel flow path. It can include a single vessel that is switched between corresponding tangential flow filtration systems to enable purification and regeneration of the resin beads using appropriate buffers while maintaining

In embodiments, regeneration of the resin beads comprises at least one low pH equilibrating vessel and an affinity-based TFF purification module configured to include both a low pH elution buffer and a regeneration buffer to enable purification, concentration and buffer exchange. This can be achieved by using at least one second tangential flow filtration system to regenerate the resin beads without the need for a separate regeneration equilibration vessel and corresponding tangential flow filtration system.

In implementations, the affinity-based TFF purification module includes at least two buffer systems.

In embodiments, the affinity-based TFF purification module includes at least one resin bead regeneration buffer system.

In embodiments, the affinity-based TFF purification module includes at least one hollow fiber membrane filter.

In embodiments, an affinity-based TFF purification module includes at least one sensor or detector.

In embodiments, an affinity-based TFF purification module includes at least one fluid handling pump.

Positive charge-based TFF purification module

In aspects, provided herein is a positive charge based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is consistent during steady state operation. and become constant

In embodiments, a positive charge based TFF purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

In embodiments, the positive charge based TFF purification module includes at least one association equilibrium vessel that allows association of a biological product with a resin bead surface; and at least one first tangential flow filtration system for separating the biological product associated resin beads from the heterogeneous mixture.

In embodiments, a positive charge based TFF purification module comprises at least one dissociation equilibrium vessel enabling dissociation of a biological product from a resin bead surface; and at least one second tangential flow filtration system for separating the resin beads from the dissociated biological product and completing their purification. In some aspects, multiple dissociation equilibrium vessels are used with multiple tangential flow filtration systems to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

In embodiments, a positive charge based TFF purification module comprises at least one regenerative equilibrium vessel; and at least one third tangential flow filtration system that allows concentration and buffer exchange of the resin beads to return the resin beads to their initial state to enable recycling and reuse of the resin beads.

In embodiments, a positive charge based TFF purification module comprises at least one collection vessel; and at least one fourth tangential flow filtration system for purifying the biological product by enabling concentration and buffer exchange of the biological product.

In embodiments, the at least one association equilibrium vessel, the at least one dissociation vessel, and the at least one regeneration equilibrium vessel of the positive charge-based TFF purification module maintain a continuous flow of filtrate through the at least one additional vessel in a parallel flow path. It may include a single vessel that is switched between corresponding tangential flow filtration systems to allow purification and regeneration of the resin beads using appropriate buffers while

In embodiments, the regeneration of the resin beads is performed in at least one second phase of a positive charge based TFF purification module configured to include at least one dissociation vessel and both a dissociation buffer and a regeneration buffer to enable purification, concentration and buffer exchange. This can be achieved by using a tangential flow filtration system to regenerate the resin beads without the need for a separate regeneration equilibrium vessel and corresponding tangential flow filtration system.

In implementations, the positive charge based TFF purification module includes at least two buffer systems.

In embodiments, the positive charge based TFF purification module includes at least one resin bead regeneration buffer system.

In embodiments, the positive charge based TFF purification module includes at least one hollow fiber membrane filter.

In embodiments, the positive charge based TFF purification module includes at least one sensor or detector.

In embodiments, the positive charge based TFF purification module includes at least one fluid handling pump.

Negative charge-based TFF purification module

In aspects, provided herein is a negative charge based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is consistent during steady state operation. and become constant

In embodiments, a negative charge based TFF purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

In embodiments, the negative charge based TFF purification module comprises at least one association equilibrium vessel enabling association of a biological product with a surface of a resin bead; and at least one first tangential flow filtration system for separating the biological product associated resin beads from the heterogeneous mixture.

In embodiments, a negative charge based TFF purification module comprises at least one dissociation equilibrium vessel that enables dissociation of a biological product from a resin bead surface; and at least one second tangential flow filtration system for separating the resin beads from the dissociated biological product and completing their purification. In some aspects, multiple dissociation equilibrium vessels are used with multiple tangential flow filtration systems to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

In embodiments, a negative charge based TFF purification module comprises at least one regeneration equilibration vessel; and at least one third tangential flow filtration system that allows concentration and buffer exchange of the resin beads to return the resin beads to their initial state to enable recycling and reuse of the resin beads.

In embodiments, a negative charge based TFF purification module comprises at least one collection vessel; and at least one fourth tangential flow filtration system for purifying the biological product by enabling concentration and buffer exchange of the biological product.

In embodiments, the at least one association equilibrium vessel, the at least one dissociation vessel, and the at least one regeneration equilibrium vessel of the negative charge based TFF purification module maintain a continuous flow of filtrate through the at least one additional vessel in a parallel flow path. It may include a single vessel that is switched between corresponding tangential flow filtration systems to allow purification and regeneration of the resin beads using appropriate buffers while

In embodiments, the regeneration of the resin beads is performed in at least one second phase of a negative charge based TFF purification module configured to include at least one dissociation vessel and both a dissociation buffer and a regeneration buffer to enable purification, concentration and buffer exchange. This can be achieved by using a tangential flow filtration system to regenerate the resin beads without the need for a separate regeneration equilibrium vessel and corresponding tangential flow filtration system.

In implementations, the negative charge based TFF purification module includes at least two buffer systems.

In embodiments, the negative charge based TFF purification module includes at least one resin bead regeneration buffer system.

In embodiments, the negative charge based TFF purification module includes at least one hollow fiber membrane filter.

In embodiments, the negative charge based TFF purification module includes at least one sensor or detector.

In embodiments, the negative charge based TFF purification module includes at least one fluid handling pump.

Isoelectric point based fluid purification module

In aspects, provided herein is an isoelectric point-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The isoelectric point-based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation.

In embodiments, a process described herein comprises continuously conveying a fraction comprising a biological product from at least one first outlet of a first module to a second module, wherein the second module undergoes free flow electrophoresis. include the device For example, a free flow electrophoresis device may include a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and an aqueous solution (e.g., an ionic solution, or a buffer or amphoteric electrolyte ( There is at least one fluid element containing a solution providing ampholyte). For example, the solution contacting surfaces of the two parallel plates include glass, ceramic, plastic, or any combination thereof. In some instances, an aqueous ionic solution may generate a pH gradient. In another example, an aqueous ionic solution may impart a constant pH.

In embodiments, a free flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module may have a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (eg, the approximate pH gradient may range from about 2 to about 10); and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separating channel (e.g., the fine pH gradient is a pH of about 5 to about 8). and at least one second fluid element including a range). For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In another embodiment, a free flow electrophoretic device includes an electric field or electric field gradient perpendicular to the direction of fluid flow and a fluidic channel created between two parallel plates to operate in a bandelectrophoresis or charge separation mode of operation, wherein the pH There is at least one fluid element with no gradient. For example, an isoelectric point-based fluid purification module includes a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7). at least one first fluid element; and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (eg, less than pH 7). .

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates to operate in an isokinetic electrophoretic mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a spacer solution (e.g. , NaCl solution) and at least one fluidic element comprising both an acidic pH gradient and a basic pH gradient.

In another embodiment, the isoelectric point-based fluid purification module includes at least one first free flow electrophoresis device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and 2 at least one second free flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, wherein each element is connected in series; can operate in an independent operating mode that enables For example, at least one first free-flow electrophoresis device may operate in an isoelectric focusing mode and at least one second free-flow electrophoresis device may operate in an isokinetic mode to increase separation resolution.

In another embodiment, an isoelectric based fluid purification module includes at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; A fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g., in the pH range of about 2 to about 10). at least one second free flow electrophoresis device comprising; and a fluid channel created between the two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. and at least one third free flow electrophoresis device. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (eg, a platinum wire electrode) to function as an anode or cathode.

In embodiments, backpressure within an isoelectric point-based fluid purification device depends on channel shape and dimensions, inlet and outlet openings and/or tubing diameters, and input flow rate. For example, the back pressure ranges from about 0.5 psi to about 10 psi. In some examples, the back pressure is controlled by, for example, without intending to limit, a needle valve.

In embodiments, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar.

In embodiments, the isoelectric point-based fluid purification device further includes an active cooling system or heat sink to enable temperature control and Joule heat dissipation. For example, an active cooling system includes an aluminum thermal chuck with a cooled circulating water/propylene glycol jacket.

In embodiments, an isoelectric point based fluid purification module includes at least one buffer or amphoteric electrolyte system.

In embodiments, an isoelectric point based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper functioning in contact with an anode or cathode, for example phosphoric acid and sodium hydroxide, respectively. In another embodiment, at least one electrode solution is configured to contact the anode or cathode to enable proper function, such as Tris buffered saline, flowing through the main separator channel, the anode channel, and the cathode channel. Contains an amphoteric electrolyte solution.

In embodiments, an isoelectric based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is arranged in-line. In some examples, the at least one sensor or detector includes, but is not limited to, a flow sensor, temperature sensor, conductivity sensor, pH sensor, refractive index detector, UV detector, or back pressure sensor.

In embodiments, an isoelectric point-based fluid purification module includes at least one liquid circuit breaker or disconnects downstream of the device and upstream of at least one in-line sensor or detector to perform sensing or detection in a voltage-free solution. make it possible

In embodiments, an isoelectric based fluid purification module includes at least one fluid handling pump.

In embodiments, an isoelectric based fluid purification module includes at least one collection vessel.

method

Provided herein are methods for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product, comprising using the processes described herein. For example, bioreactor types include, but are not limited to, batch bioreactors, fed-batch bioreactors, perfusion bioreactors, chemostat bioreactors, or multi-compartment bioreactors. In some instances, a bioreactor produces the biological product at steady state.

In embodiments, this specification includes, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or isoelectric point A method for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product is provided, comprising utilizing at least one of the modules described herein, such as a fluid purification module based on a substrate.

In some embodiments, the present specification includes, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or isoelectric point A method for continuously purifying a biological product from a heterogeneous mixture derived from a bioreactor that produces the biological product at steady state comprising using at least one of the modules described herein, such as a fluid purification module based on Provided.

In another embodiment, the present specification includes, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or isoelectric point Provided is a method of purifying a biological product from a heterogeneous mixture that is not derived from a bioreactor that produces the biological product at steady state, comprising using at least one of the modules described herein, such as a fluid purification module based on do.

Other aspects of the invention are disclosed below.

This patent or application file contains one or more drawings made in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
1A-1D depict schematic diagrams of exemplary continuous process flows described herein. 1A shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based magnetic purification. module, step 4 includes at least one charge-based magnetic purification module, step 5 includes a standard industrial virus inactivation and filtration process, eg carried out in a fed-batch or perfusion mode, 6 includes, for example, high performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of step 7. 1B shows an exemplary continuous process flow, wherein step 1 includes a bioreactor producing biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based magnetic purification module. Step 4 includes a positive charge-based magnetic purification module, step 5 includes a negative charge-based magnetic purification module, and step 6 includes, for example, preparing the standard industrial charge-finish process of step 7. high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode for 1C shows an exemplary continuous process flow, wherein step 1 includes a bioreactor producing biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based magnetic purification. module, step 4 includes an isoelectric point based fluid purification module, step 5 includes a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 includes a step 7 includes high-performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for standard industrial fill-finish processes. 1D shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based magnetic purification. module, step 4 includes an isoelectric based fluid purification module, step 5 is high performance tangential flow using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of step 6 Include filtration.
2A-2D show schematic diagrams of exemplary continuous process flows described herein. 2A shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based purification module. step 4 includes at least one charge-based purification module, step 5 includes a standard industrial virus inactivation and filtration process, e.g. performed in a fed-batch or perfusion mode, and step 6 includes e.g. For example, step 7 includes high performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for standard industrial fill-finish processes. 2B shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based purification module. step 4 includes a positive charge-based purification module, step 5 includes a negative charge-based purification module, and step 6 includes, for example, fed-batch or flow-through to prepare for a standard industrial charge-finish process of step 7. It includes high performance tangential flow filtration using charged membranes performed in the mode. 2C shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based purification module. Step 4 includes an isoelectric point based fluid purification module, Step 5 includes a standard industrial virus inactivation and filtration process, eg performed in a fed-batch or perfusion mode, and Step 6 includes the steps of step 7. It includes high-performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for standard industrial fill-finish processes. 2D shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based purification module. Step 4 includes an isoelectric based fluid purification module, and Step 5 includes high performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of Step 6. include
3A-3D show schematic diagrams of exemplary continuous process flows described herein. 3A shows an exemplary continuous process flow, wherein step 1 includes a bioreactor producing biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based fluid purification. module, step 4 includes at least one charge-based fluid purification module, step 5 includes a standard industrial virus inactivation and filtration process, eg performed in a fed-batch or perfusion mode, 6 includes, for example, high performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of step 7. 3B shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based fluid purification. module, step 4 includes a positive charge-based fluid purification module, step 5 includes a negative charge-based fluid purification module, and step 6 prepares, for example, a standard industrial charge-finish process of step 7. high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to 3C shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based fluid purification. module, step 4 includes an isoelectric point based fluid purification module, step 5 includes a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 includes a step 7 includes high-performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for standard industrial fill-finish processes. 3D shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes affinity-based fluid purification. module, step 4 includes an isoelectric based fluid purification module, step 5 is high performance tangential flow using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of step 6 Include filtration.
4A-4D show schematic diagrams of exemplary continuous process flows described herein. 4A shows an exemplary continuous process flow, wherein step 1 includes a bioreactor that produces biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based TFF purification module. wherein step 4 includes at least one charge-based TFF purification module, step 5 includes a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 includes , for example, high performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of step 7. 4B shows an exemplary continuous process flow, wherein step 1 includes a bioreactor producing biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based TFF purification module. Step 4 includes a positive charge-based TFF purification module, Step 5 includes a negative charge-based TFF purification module, and Step 6 includes, for example, a cost-effective process to prepare for the standard industrial charge-finish process of Step 7. It includes high performance tangential flow filtration using charged membranes performed in feed or perfusion mode. 4C shows an exemplary continuous process flow, wherein step 1 includes a bioreactor producing biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based TFF purification module. wherein step 4 includes an isoelectric point based fluid purification module, step 5 includes a standard industrial virus inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes step 7 of high performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for standard industrial fill-finish processes. 4D shows an exemplary continuous process flow, wherein step 1 includes a bioreactor producing biological products at steady state, step 2 includes a continuous dynamic filtration module, and step 3 includes an affinity-based TFF purification module. wherein step 4 includes an isoelectric point-based fluid purification module, and step 5 includes high-performance tangential flow filtration using charged membranes performed in fed-batch or once-through mode to prepare for the standard industrial fill-finish process of step 6. includes
5A and 5B show exemplary continuous process flows along with design schematics for the downstream purification modules described herein.
6A and 6B show a series of dynamic filtration devices comprising a single output head for continuously delivering a heterogeneous mixture comprising biological products from a steady state bioreactor output line, and a separate output head for supplying wash buffer. represents an exemplary design of 6A shows a single output head for continuously delivering a heterogeneous mixture comprising biological products from a steady state bioreactor output line; separate output head for supplying wash buffer; a rolling filter membrane functioning as a supply reel, a collection reel; Two servo motors to control the supply reel-to-collect reel system; two support rods with mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and having an opening continuous with the vacuum line; vacuum collection vessel; diaphragm pump; and a peristaltic pump; a schematic diagram of a dynamic filtration device design. 6B shows a single output head for continuously delivering a heterogeneous mixture comprising biological products from a steady state bioreactor output line; separate output head for supplying wash buffer; a rolling filter membrane functioning as a supply reel, a collection reel; Two servo motors to control the supply reel-to-collect reel system; two support rods with mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and having an opening continuous with the vacuum line; controllable T-valve; two vacuum collection vessels; diaphragm pump; and two peristaltic pumps.
7A and 7B show dynamic filtration comprising multiple output heads for continuously delivering a heterogeneous mixture comprising biological products from a steady state bioreactor output line, and multiple separate output heads for supplying wash buffer. Two exemplary designs of the device are shown. 7A shows multiple output heads for conveying a heterogeneous mixture comprising biological products from a steady state bioreactor output line; a plurality of individual output heads for supplying wash buffer; a rolling filter membrane functioning as a supply reel, a collection reel; Two servo motors to control the supply reel-to-collect reel system; two support rods with mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and having an opening continuous with the vacuum line; vacuum collection vessel; diaphragm pump; and a peristaltic pump; a schematic diagram of a dynamic filtration device design is shown. 7B shows multiple output heads for continuously delivering a heterogeneous mixture comprising biological products from a steady state bioreactor output line; a plurality of individual output heads for supplying wash buffer; a rolling filter membrane functioning as a supply reel, a collection reel; Two servo motors to control the supply reel-to-collect reel system; two support rods with mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and having an opening continuous with the vacuum line; controllable T-valve; two vacuum collection vessels; diaphragm pump; and two peristaltic pumps.
8 shows an image of an exemplary membrane support structure having an opening with five parallel slots.
9A and 9B are images showing the removal of PolyBeads (0.05% solids; 2 μm (red), 6 μm (red), and 10 μm (blue) diameter) from a heterogeneous mixture in IX PBS. 9A is an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameter) in 1X PBS; filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-3 represents a sample dynamically filtered with a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL/min); and a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-4 represents samples dynamically filtered with a 0.45 μm PES filter membrane and flow rates of 0.25, 0.5, 1.0, 2.0 and 5.0 mL/min); An image showing a visual comparison (from left to right) of 9B is an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameter) in 1X PBS; filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device with a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL/min (MBM-3); and a filtrate (MBM-4) obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device with a 0.45 μm PES filter membrane and flow rates of 0.25, 0.5, 1.0, 2.0, and 5.0 mL/min; As a bar graph showing , which demonstrates the successful removal of PolyBeads from a heterogeneous mixture using the exemplary dynamic filtration device.
10A to 10D are samples of PolyBeads (0.05% solids; 2 μm (red), 6 μm (red), and 10 μm (blue) diameter) from a heterogeneous mixture of PolyBeads suspended in a 0.5 mg/mL solution of BSA-FITC in 1X PBS. A series of data showing the removal. 10A is a UV-Vis spectrophotometric trace of a serially diluted heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameter) suspended in a 0.5 mg/mL solution of BSA-FITC in 1X PBS. As a graph showing, the PolyBead signature area is displayed, indicating that PolyBeads exist. 10B is a graph showing UV-Vis spectrophotometer traces of serially diluted 0.5 mg/mL solutions of BSA-FITC in 1X PBS, with PolyBead signature regions marked and no PolyBeads. 10C shows an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm and 10 μm diameter) suspended in a 0.5 mg/mL solution of BSA-FITC in 1X PBS; filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-3a represents a sample dynamically filtered with a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL/min); a filtrate obtained by purification of the heterogeneous mixture with an exemplary dynamic filtration device (MBM-4a represents a sample dynamically filtered with a 0.45 μm PES filter membrane and a flow rate of 0.5 mL/min); a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-5a represents a sample dynamically filtered with a 0.45 μm PES filter membrane and a flow rate of 2.0 mL/min); and supernatant collected from purification of the heterogeneous mixture by centrifugation (5 min at 10,000xg); images are shown for visual comparison (left to right). 10D shows an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameter) suspended in a 0.5 mg/mL solution of BSA-FITC in 1X PBS; filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration apparatus (MBM-3a); a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration apparatus (MBM-4a); a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration apparatus (MBM-5a); and UV-Vis spectrophotometric comparison of supernatants collected from purification by centrifugation (5 min at 10,000xg), using an exemplary dynamic filtration device, as indicated by the absence of PolyBeads in the PolyBead signature region. , demonstrating that BSA-FITC is successfully purified by removing the PolyBeads.
11A and 11B show dynamic filtration of a heterogeneous mixture of PolyBeads suspended in a 0.5 mg/mL solution of human polyclonal IgG (hlgG) in 1X PBS at an input flow rate of 10 mL/min. 11A shows dynamic filtration through a slot die output head. 11A shows an initial heterogeneous mixture of PolyBeads (2 μm, 6 μm, and 10 μm diameter at 1.1×10 ⁸ , 4.2×10 ⁶ , and 1.0×10 ⁶ particles/mL, respectively) suspended in a 0.5 mg/mL solution of hIgG in 1X PBS; a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device with a 0.45 μm PES filter membrane and a flow rate of 10 mL/min; and supernatant collected from purification of the heterogeneous mixture by centrifugation (5 min at 10,000xg); images are shown for visual comparison (left to right). 11B is a graph showing a spectrophotometric comparison of total protein concentration determined by BCA assay of filtrate obtained by dynamic filtration, and supernatant collected by centrifugation. 11C shows PolyBeads (7.3x10 ⁷ , 1.1x10 ⁸ , 1.1x10 ⁸ , 1.1x10 ⁸ , 3.4x10 ⁷ , and 1.0x10 ⁶ particles/mL, respectively) suspended in a 0.5 mg/mL solution of hIgG in 1X PBS. , 0.75 μm, 1 μm, 2 μm, 3 μm, and 10 μm diameter) of the initial heterogeneous mixture; filtrate obtained from purification of the heterogeneous mixture with an exemplary dynamic filtration device with a 0.45 μm PES filter membrane and a flow rate of 10 mL/min, and supernatant collected from purification of the heterogeneous mixture by centrifugation (5 min at 10,000xg); Displays images for visual comparison (from left to right). 11D is a graph showing a spectrophotometric comparison of total protein concentration determined by BCA assay of filtrate obtained by dynamic filtration, and supernatant collected by centrifugation.
12A-12D show protein recovery of dynamic filtration for different proteins, protein concentrations, filter membrane materials and pore sizes, and membrane support structures, and during continuous operation at different input flow rates. Figure 12a shows proteins of different sizes and charges (0.5-10 mg/mL, respectively) by BCA assay of the filtrate obtained by dynamic filtration with a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec and an input flow rate of 10 mL/min. , 5 mg/mL, and 0.5 mg/mL of bovine serum albumin (BSA), lysozyme, and hIgG). Figure 12b shows the filtrate obtained by dynamic filtration using filter membranes of different materials and pore sizes (0.45 μm PES, 0.45 μm hydrophilic PVDF, 0.22 μm PES) with a transport rate of 0.5 mm/sec and an input flow rate of 10 mL/min. Recovery of 0.5 mg/mL of hIgG by BCA assay is shown. 12C shows a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec and an input flow rate of 10 mL/min, and different membrane support structures (PTFE membrane support structure with 5 parallel slots, and porous hydrophilic polyethylene (PE)). The recovery of hIgG at 0.5 mg/mL by BCA assay of the filtrate obtained by dynamic filtration using a PTFE membrane support structure with an insert) is shown. Figure 12d shows lysozyme at 0.5 mg/mL by BCA assay of filtrates obtained by dynamic filtration at different flow rates (5 and 10 mL/min) during long-term continuous operation using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec. represents the recovery rate of
Figures 13A-13C compare cell purification by dynamic filtration and centrifugation of an input heterogeneous mixture comprising a suspension cell culture in RPMI medium spiked with hIgG to a final concentration of 1 g/L. FIG. 13A shows an initial hlgG spiked murine myeloma suspension cell culture in RPMI medium (lg/L hIgG at 2×10 ⁶ cells/mL); Filtrates (DF-1, DF-2, DF-3) obtained by dynamic filtration at an input flow rate of 2 mL/min using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec; and supernatants (C-1, C-2, C-3) collected by centrifugation at 10,000xg for 5 min; FIG. 13B shows an initial hlgG spiked murine myeloma suspension cell culture in RPMI medium (lg/L hIgG at 2×10 ⁶ cells/mL); Filtrates (DF-1, DF-2, DF-3) obtained by dynamic filtration at an input flow rate of 2 mL/min using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec; and SDS-PAGE analysis (from left to right) of supernatants (C-1, C-2, C-3) obtained by centrifugation at 10,000xg for 5 minutes. 13C shows the filtrate obtained by dynamic filtration at an input flow rate of 2 mL/min using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec (blue outline bar); and comparison of hIgG recovery from a heterogeneous mixture (suspension cell culture in RPMI medium spiked with 1 g/L hIgG) by BCA assay of supernatants collected by centrifugation at 10,000xg for 5 minutes.
14 shows an exemplary design schematic of an affinity-based magnetic purification device comprising a loop conveyor system and at least one magnetic field that is permanently “on”.
15 shows an exemplary design schematic of an affinity-based magnetic purification device comprising a loop conveyor system and at least one magnetic field capable of “on/off” toggling.
16 shows an exemplary design schematic of a charge-based magnetic purification device comprising a loop conveyor system and at least one magnetic field that is permanently “on”.
17 shows an exemplary design schematic of a charge-based magnetic purification device comprising a loop conveyor system and at least one magnetic field switchable “on/off”.
18 shows an exemplary design schematic of an affinity-based magnetic refining device comprising a pick-and-place robotic system and at least one magnetic field.
19 shows an exemplary design schematic of a charge-based magnetic purification device comprising a pick-and-place robotic system and at least one magnetic field.
20A and 20B show a mixture of hIgG (target, 2 g/L input concentration) and lysozyme (impurity, 1 g/L input concentration) performed with an affinity-based magnetic purification device packed with magnetic protein A coated agarose beads. affinity-based magnetic purification. Figure 20A shows total protein analysis via BCA assay on fractions collected from three consecutive cycles of magnetic affinity bead use and recycling, which reproducibly allow magnetic affinity beads to occur without compromising binding capacity and performance. Demonstrate ability to be recycled and reused. 20B shows SDS-PAGE analysis of fractions collected from three consecutive cycles of magnetic affinity bead use and recycling, which showed that magnetic affinity beads were reproducibly recycled and reused without compromising binding capacity and performance. prove your ability to be
21A-21D show an exemplary design of an affinity-based purification device comprising a mechanical rotation system. 21A is a schematic diagram of a lid system. 21B is a schematic diagram of a container carousel. 21C is a schematic diagram of a collection system. 21D shows how the lid system and collection system interface with the container carousel.
22A-22D show an exemplary design of a charge-based purification device that includes a mechanical rotation system. 22A is a schematic diagram of a lid system. 22B is a schematic diagram of a container carousel. 22C is a schematic diagram of a collection system. 22D shows how the lid system and collection system interface with the container carousel.
23A to 23D show individual system components of an affinity-based purification or charge-based purification device. 23A is a schematic diagram of an exemplary gasket lid, container, and collector assembly. 23B is a schematic diagram of an exemplary gasket lid including an air inlet, two buffer inlets configured to create a circular flow pattern, a vent port, and a fill inlet, which are components of the lid system. 23C is a schematic diagram of a vessel including a mesh filter or frit, which is a component of a vessel carousel, and a valve. 23D is a schematic diagram of a collector that is a component of the collection system.
24A and 24B show an exemplary design of an affinity-based purification apparatus comprising a stepwise linear system. 24A shows the individual system components of an affinity-based purification device. 24b shows the connectivity of an affinity-based purification device comprising a stepwise linear system.
25A and 25B show an exemplary design of an affinity-based purification device comprising a stepwise linear system. 25A shows the individual system components of a charge based purification device. 25B shows the connectivity of a charge-based purification device comprising a stepwise linear system.
26A and 26B show affinity-based purification of a hIgG (2 g/L input concentration) solution performed with an affinity-based purification apparatus filled with protein A coated agarose resin beads. Figure 26A shows total protein analysis via BCA assay on fractions collected from three consecutive cycles of affinity resin bead use and recycling, which reproducibly allows affinity resin beads to be used without compromising binding capacity and performance. Demonstrate ability to be recycled and reused. 26B shows SDS-PAGE analysis of fractions collected from three consecutive cycles of affinity resin bead use and recycling, which showed that affinity resin beads were reproducibly recycled and reused without compromising binding capacity and performance. prove your ability to be
27A and 27B show a mixture of hIgG (target, 2 g/L input concentration) and lysozyme (impurity, 1 g/L input concentration) performed with an affinity-based purification device packed with protein A coated agarose resin beads. Represents affinity-based purification. 27A shows total protein analysis via BCA assay on fractions collected from three consecutive cycles of affinity resin bead use and recycling, which reproducibly allow affinity resin beads to be used without compromising binding capacity and performance. Demonstrate ability to be recycled and reused. 27B shows SDS-PAGE analysis of fractions collected from three consecutive cycles of affinity resin bead use and recycling, which showed that affinity resin beads were reproducibly recycled and reused without compromising binding capacity and performance. prove your ability to be
28A-28D are a series of images showing exemplary designs of hybrid fluid elements. 28A is an image showing a hybrid fluidic device comprising parallel cross-flow channels, a permanent magnetic field, and two piezoelectric transducers. 28B is an image showing a hybrid fluidic element comprising an angled cross-flow channel, a permanent magnetic field, and two piezoelectric transducers. 28C is an image showing a hybrid fluidic device comprising parallel cross-flow channels, a permanent magnetic field, and two optional dielectrophoretic electrodes. 28D is an image showing a hybrid fluidic device comprising an angled cross-flow channel, a permanent magnetic field, and two optional dielectrophoretic electrodes.
29 shows an exemplary design schematic of an affinity-based fluid purification device.
30 shows an exemplary design schematic of a charge-based fluid purification device.
31 shows an exemplary design schematic of an affinity-based purification device comprising at least one tangential flow filtration system.
32 shows an exemplary design schematic of a charge-based purification device comprising at least one tangential flow filtration system.
33 shows an exemplary design schematic of an isoelectric based fluid purification device comprising a fluid element with a channel created between two parallel plates, an electric field perpendicular to the direction of fluid flow, and an aqueous ionic solution.
34 shows a channel created between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a second fluid element having a fine pH gradient, a channel created between two parallel plates, fluid flow An exemplary design schematic of a free flow electrophoretic device comprising a first fluid element with an electric field perpendicular to the direction and an approximate pH gradient, wherein the device can operate in an isoelectric focusing mode.
35 shows a channel created between the two parallel plates, an electric field perpendicular to the direction of fluid flow, and a second fluid element with constant acidic pH across the main separating channel, connected to the second fluid element. An exemplary design schematic of a free flow electrophoretic device comprising a first fluidic element having a created channel, an electric field perpendicular to the direction of fluid flow, and a constant basic pH across a main separating channel, the device comprising a belt electrophoretic device. It can operate in electrophoresis mode.
36 shows a channel created between two parallel plates that can operate in isokinetic electrophoresis mode and a second fluid element that has an electric field perpendicular to the direction of fluid flow, connected to a second fluid element that can operate in isoelectric focusing mode. An exemplary design schematic of a free flow electrophoretic device including a channel created between two parallel plates and a first fluidic element having an electric field orthogonal to the direction of fluid flow is shown.
37 shows a channel created between two parallel plates that can operate in isokinetic electrophoresis mode and a third fluidic element with an electric field perpendicular to the direction of fluid flow, connected to two channels that can operate in isoelectric focusing mode. a first fluid element having a channel created between two parallel plates and a channel with an optional dielectrophoretic electrode for pre-classifying a mixture connected to a second fluid element having an electric field perpendicular to the direction of fluid flow; An exemplary design schematic of a free flow electrophoresis device is shown.
FIG. 38 shows the design of an exemplary bubble removal and degassing system that removes electrolytic bubbles directly from the electrode channels to create a bubble-free primary isolation channel.
39 shows an exemplary liquid circuit breaker that creates a disconnect to a solution connected to an applied voltage flowing from the outlet of a free flow electrophoretic device to at least one in-line sensor or detector.
40a to 40e show rhodamine 6G using an isoelectric point-based purification apparatus having an anode channel (H ₂ SO ₄ ), a cathode channel (NaOH), and a main separation channel with five inlets and five outlets through which the ampholyte solution flows. (Rhodamine 6G) (0.25mg/mL, net charge +1) and Fluorescein (0.25mg/mL, net charge -1) isoelectric point-based fluid purification of a mixture, wherein the mixture is the device's It was introduced at the center of the inlet (Inlet 3). 40A shows optical images of fractions collected from 5 outlets at 0 V and 5 mL/min. Figure 40b shows absorbance spectra of fractions collected from five outlets at 0 V and 5 mL/min. 40C shows optical images of fractions collected from 5 outlets at 1000 V and 10 mL/min in the presence of a pH gradient. 40D shows absorbance spectra of fractions collected from 5 outlets at 1000 V and 10 mL/min in the presence of a pH gradient. Figure 40E shows the purification of the mixture resulting in fractions comprising purified rhodamine 6G (outlet 2, towards the cathode) and purified fluorescein (outlet 4, towards the anode).
41A to 41C show different operations using an isoelectric based purification device with an anode channel (H ₂ SO ₄ ), a cathode channel (NaOH), and a main separation channel with 5 inlets and 5 outlets through which the ampholyte solution flows. Optical imaging is shown for purifying a mixture of rhodamine 6G (0.25 mg/mL, net charge +1) and fluorescein (0.25 mg/mL, net charge -1) under conditions wherein the mixture is in the device was introduced at the center of the inlet (inlet 3). 41A shows isoelectric focusing free flow electrophoresis of a mixture of Rhodamine 6G and Fluorescein at 500 V and a flow rate of 3 mL/min. 41B shows isoelectric focusing free flow electrophoresis of a mixture of Rhodamine 6G and Fluorescein at 700 V and a flow rate of 5 mL/min. 41C shows isoelectric focusing free flow electrophoresis of a mixture of Rhodamine 6G and Fluorescein at 900 V and a flow rate of 10 mL/min.
42A and 42B show optical imaging of the purification of a mixture of small molecule dyes using an isoelectric point-based purification device, which includes an anode channel (H ₂ SO ₄ ), a cathode channel (NaOH), and an amphoteric electrolyte solution. It includes a main separation channel with 5 inlets and 5 outlets flowing through which the mixture is introduced at the center of the inlet (inlet 3) of the device. 42A isoelectric focusing free flow electrophoresis of a mixture of Basic Fuchsin (0.05 mg/mL, net charge +3) and Fluorescein (0.25 mg/mL, net charge -1) at 500 V and flow rate 5 mL/min. indicates 42b isoelectric focusing free flow electrophoresis of a mixture of Crystal Violet (0.05 mg/mL, net charge +3) and fluorescein (0.25 mg/mL, net charge -1) at 500 V and flow rate 5 mL/min. indicates
43A to 43D show basic fuchsine (0.005 mg/mL, net charge +3) and fluorescein (0.25 mg/mL) using an isoelectric point based purification apparatus operating in isoelectric focusing free flow electrophoresis mode over increasing applied voltages. , showing optical imaging on purifying a mixture of net charge −1). The mixture was introduced into the central inlet (inlet 3) at a flow rate of 5 mL/min using an apparatus comprising an anode channel, a cathode channel, and a main separation channel with 5 inlets and 10 outlets, each channel having the same The amphoteric electrolyte solution was flowed at 5 mL/min. When no voltage is applied, the mixture exits the device at the central outlet (outlets 4 and 5) following a laminar flow (FIG. 43a). When voltage is applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 5 mL/min, a linear pH gradient is established, and basic fuchsin and fluorescein migrate to the cathode and anode, respectively, which correspond to the theoretical electrophoretic mobility Consistent with predictions (FIGS. 43B-43D). As the electric field strength was increased by increasing the applied voltage from 600 V (FIG. 43B) to 900 V (FIG. 43C) and 1100 V (FIG. 43D), the separation of the two molecules was observed to increase proportionally to the length of the main separation channel.
44A and 44B show optical imaging of the purification of a mixture of small molecule dyes using an isoelectric point-based purification device, which is separated by an anode channel (H ₂ SO ₄ ), a cathode channel (NaOH), and a spacer solution. and a main separating channel having 5 inlets and 5 outlets through which the two amphoteric electrolyte solutions flowed, wherein the mixture was introduced into the spacer solution at the center of the inlet (inlet 3) of the device. 44A shows isokinetic electrophoresis of a mixture of rhodamine 6G (0.25 mg/mL, net charge +1) and fluorescein (0.25 mg/mL, net charge -1) at 250 V and flow rate 5 mL/min, resulting in The two dyes are concentrated into two separate high-resolution lines. FIG. 44B shows the UV irradiation results of the results shown in FIG. 44A.
45a to 45e show the results of purifying a mixture of BSA (0.5mg/mL, pI 4-5) and lysozyme (0.25mg/mL, pI 11) using an isoelectric point-based purification device, which is a bipolar channel ( H ₂ SO ₄ ), a cathode channel (NaOH), and a main separation channel having 5 inlets and 5 outlets through which the ampholyte solution flows, wherein the mixture is introduced centrally at the inlet (inlet 3) of the device. It became. 45A shows spectrophotometric analysis by BCA assay of the total protein concentration of fractions from 5 outlets at 0 V and a flow rate of 10 mL/min. 45B shows SDS-PAGE analysis of fractions from 5 outlets at 0 V and flow rate 10 mL/min, showing that a mixture is present in outlet 3. 45C shows a spectrophotometric analysis by BCA assay of the total protein concentration of fractions from 5 outlets at 850 V and a flow rate of 10 mL/min, showing that protein is distributed over outlets 2, 3 and 4. 45D shows SDS-PAGE analysis of fractions from 5 outlets at 850 V and flow rate 10 mL/min, showing that purified lysozyme is present in outlet 2 and purified BSA is present in outlet 4. 45E shows the theoretical electrophoretic migration directions of BSA (towards the anode) and lysozyme (towards the cathode).
46a to 46c show the results of purifying a mixture of hlgG (0.5 mg/mL, pl 6-8) and lysozyme (0.25 mg/mL, pl 11) using an isoelectric point-based purification device, which has a bipolar channel ( H ₂ SO ₄ ), a cathode channel (NaOH), and a main separation channel having 5 inlets and 5 outlets through which the ampholyte solution flows, wherein the mixture is introduced at the center of the inlet (inlet 3) of the device. It became. 46A shows a voltage at (1) 0 V, 5 mL/min, (2) 1000 V, 5 mL/min, (3) 1500 V, 5 mL/min, (4) 0 V, 10 mL/min, or (5) 5 at 1000 V, 10 mL/min. Spectrophotometric analysis by BCA assay of the total protein concentration of fractions from canine effluent is shown. 46B shows the theoretical electrophoretic migration directions of hIgG (toward the anode, cathode, and center) and lysozyme (towards the cathode). FIG. 46C shows 5 at (1) 0 V, 5 mL/min, (2) 1000 V, 5 mL/min, (3) 1500 V, 5 mL/min, (4) 0 V, 10 mL/min, or (5) 1000 V, 10 mL/min. SDS-PAGE analysis of fractions from canine effluent is shown.
47a to 47d show the results of purifying a mixture of BSA (0.5mg/mL, pI 4-5) and lysozyme (0.25mg/mL, pI 11) using an isoelectric point-based purification device, which is a bipolar channel ( H ₂ SO ₄ ), a cathode channel (NaOH), and a main separation channel having 5 inlets and 5 outlets through which the ampholyte solution flows, wherein the mixture is introduced centrally at the inlet (inlet 3) of the device. It became. 47A shows a spectrophotometric analysis by BCA assay of the total protein concentration of fractions from five outlets at 0 V and 500 V at a flow rate of 3 mL/min, showing protein at outlets 2, 3, and 4 under applied voltage. shows that it is distributed over 47B shows spectrophotometric analysis by BCA assay of the total protein concentration of fractions from 5 outlets at 0 V and 700 V at a flow rate of 5 mL/min, showing protein at outlets 2, 3, and 4 under applied voltage. shows that it is distributed over 47C shows a spectrophotometric analysis by BCA assay of the total protein concentration of fractions from 5 outlets at 0 V and 850 V at a flow rate of 10 mL/min, with protein under applied voltage across outlets 2, 3, and 4 shows that it is distributed. 47D shows SDS-PAGE analysis of fractions from five outlets at (1) 0 V or 500 V at 3 mL/min, (2) 0 V or 700 V at 5 mL/min, or (3) 0 V or 850 V at 10 mL/min. indicate
48 is a charge based purification module, magnetic purification module, charge based purification module in an exemplary semi-continuous process described herein using standard industrial downstream processing equipment running in a fed-batch or once-through mode to prepare a biological product for charge finishing. Exemplary schematics for connecting purification modules, charge-based fluid purification modules, charge-based TFF purification modules, or isoelectric point-based purification modules are shown.
49 is an exemplary semi-continuous process described herein using standard industrial downstream processing equipment running in fed-batch or perfusion mode to prepare biological products for fill finishing in the absence of independent viral inactivation and removal process steps. shows an exemplary schematic for connecting a charge-based purification module, a magnetic purification module, a charge-based purification module, a charge-based fluid purification module, a charge-based TFF purification module, or an isoelectric point-based purification module.

In this specification, in particular, a continuous process for purifying a biological product is provided. The presently claimed process includes, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses, or lentiviruses, such as It offers many advantages over current downstream methods and processes for purifying biological products. For example, without intending to be limiting, the processes described herein include an initial filtration comprising at least one dynamic filtration module as described herein to remove large impurities (eg, cells, cell debris, and aggregates). Provided is a continuous bioprocess for purifying monoclonal antibodies that eliminates the problem of membrane fouling inherent in conventional multi-stage filtration processes (eg, multi-stage tangential flow filtration or depth filtration). In addition, the continuous process reduces production facility footprint, time required for facility construction and validation, facility build-up and Significantly reduce associated costs, and capital equipment expenditures.

Continuous bioprocessing as described herein, due to its ability to operate continuously, requires large process equipment - the size of which is large bioreactors - for the centrifugation, depth filtration, and column chromatography steps of existing downstream bioprocessing. Volume dependent - allows for the use of smaller and streamlined equipment (eg, smaller bioreactor volume and downstream bioprocess equipment) since no volume dependent is required. Additionally, smaller and streamlined equipment that operates continuously allows the use of much smaller bioreactor(s) that produce monoclonal antibodies at steady state. A continuous bioprocess as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and biological product loss when compared to existing monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems having a footprint that occupies significantly fewer square feet than current technologies without sacrificing product throughput or yield on a kilograms per year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require at least 200,000 square feet.

A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based magnetic purification module, and a charge-based magnetic purification module or an isoelectric point-based fluid purification module.

A continuous process for purifying a biological product is described, said process comprising continuously receiving through an input line a heterogeneous mixture comprising the biological product, wherein the biological product is a protein or fragment thereof (polypeptide), an antibody or fragments thereof, cytokines, chemokines, enzymes, or growth factors. Upon purification, biological products (e.g., monoclonal antibodies) are free from impurities (cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts). , buffer components, surfactants, sugars, metal contaminants, leachates, media components, and/or naturally occurring organic molecules with which they are naturally associated) at least 60%, 70%, 80%, 90%, It is substantially pure when 95% by weight, or even 99% by weight is removed.

The process involves the continuous removal of large impurities from the heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module generating a filtrate comprising biological product by continuously supplying biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure. The dynamic filtration module may further include at least one additional input line for supplying the wash buffer through a coaxial output head or a separate monoaxial output head.

In embodiments, the processes described herein include purifying biological products produced continuously in bioreactors (eg, fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, a bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, the output bleed line providing continuous fluid flow from the bioreactor to the dynamic filtration module. It serves as an input line allowing flow.

As described herein, processes for continuously removing large impurities from a heterogeneous mixture (or mixture) include centrifugation, disk-stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any of these. do not include combinations The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, a filter membrane (or depth filter) has a finite capacity and when the membrane reaches its capacity, it is filtered. The rate decreases (eg membrane pores become clogged). In "static" (as opposed to "dynamic") filtration, the filter membrane remains stationary (does not move) and a flow (eg, flow of a heterogeneous mixture) passes through a stationary filter membrane. Such static filtration methods are common in the art, simple and well known.

Unlike the static filtration methods commonly used in the art, the process herein describes a dynamic filtration module, wherein the components of a dynamic filtration module allow filtration to continuously occur in new, unused target areas of the filter membrane. (e.g., the membrane moves or advances depending on the flow rate of the overall process). This prevents membrane fouling or clogging and allows control of filter cake packing and thickness during operation.

The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, a vacuum line, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll comprises a rolled filter membrane, wherein the filter membrane is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF , polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

The pore size of the rolled filter membrane depends on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

The filter membrane roll has a width of about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system or membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts with a prefabricated roll and continues to an initially empty collection roll to create a reel-to-reel system. In aspects, a dynamic filtration module includes a rolled filter membrane extending between a supply reel and a collection reel, the filter membrane having a target area (eg, an active target area) configured to receive a heterogeneous mixture. For example, feed reel movement is controlled by a servo motor coupled with a gearbox that limits revolutions per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collecting reel movement is controlled by a servo motor coupled with a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. Additionally, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure consistent speed with the constantly changing diameter of the filter membrane rolls on both the feed and collect reels during operation. For example, the supply reel and the collection reel run in the same direction at the same speed.

In embodiments, the transport rate of the filter membrane ranges from about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1 mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includes a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg PTFE), and an opening continuous with the vacuum line. As used herein, “membrane support structure” refers to a component constructed to provide structural support to the active area of a filter membrane to prevent deformation as it passes through a negative pressure area due to an opening continuous with the vacuum line. Also, as used herein, a “mechanically smooth contact surface” refers to a surface that has a low static coefficient of friction, particularly when wet, that creates a low frictional force opposing the transport of the filter membrane. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. Mechanically flat contact surfaces can also be measured by surface roughness, the lower the value the smoother the surface. Also, because rough surfaces have greater friction between surfaces than smooth surfaces, mechanically smooth contact surfaces as used herein refer to surfaces with low friction (i.e., low static coefficient of friction).

In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (eg, a mesh, at least one slot, at least one hole, or any combination thereof). The opening may also include regularly spaced elements, for example, the opening may include a series of equally spaced parallel slots. Additionally, the opening may comprise a grate (eg, a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, where each grate is vertical. The opening may be a collection of irregular or regular elements (eg a series of parallel slots). The openings may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (eg, mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without intending to be limiting, the openings may include a mesh between about 0.25 mm and about 5 mm thick.

The membrane support structure of the dynamic filtration module contains a temperature control mechanism. The temperature control mechanism maintains the temperature from about 4° C. to about 37° C. when there is evaporative cooling. For example, during antibody purification, a temperature control mechanism maintains a temperature between 15°C and 37°C. Exemplary temperature control mechanisms include, but are not limited to, single loop controllers, multiple loop controllers, closed loop controllers, PID controllers, Peltier elements, resistive heating elements, and/or thermal chucks with circulating water/propylene glycol jackets.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, PTFE, PFA). For example, the static coefficient of friction ranges from about 0.01 to about 0.1, from about 0.01 to about 0.05, or from about 0.05 to about 0.1. In a specific example, the static friction coefficient is 0.04. For example, the dynamic filtration module includes at least one support rod or roller with a mechanically flat contact surface to stabilize the movement of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to a target area (eg, an active target area) of the filter membrane. For example, the at least one output head is a tube or slot die.

In some embodiments, the dynamic filtration module includes at least one additional input line for supplying wash buffer through a coaxial output head, a separate monoaxial output head, a separate slot die output head, or a slot die output head with multiple openings. additionally includes

In some embodiments, the dynamic filtration module may include elements known in the coatings and converting industry, such as, but not intended to be limiting, active or passive edge guides, tension regulators (eg, dancers), brakes and tension detectors; or any combination thereof.

In embodiments, a dynamic filtration module includes a vacuum system in continuity with a membrane support structure to apply a negative pressure across an active target area of a filter membrane, wherein the negative pressure causes active transport of the filter membrane across the membrane support structure. and allow collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about 0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. In aspects described herein, during purification by dynamic filtration, the filtrate comprising biological products is fed under negative pressure to a vacuum collection vessel capable of collecting about 50 mL to about 100 L. For example, a vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 50 L.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process includes a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced.

The process described herein involves continuously passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product. For example, separating a solution into two or more fractions may include at least one fraction comprising biological products, and at least one other fraction comprising minor impurities. As described herein, the first module includes an affinity-based magnetic purification device. "Affinity-based magnetic purification device" refers to a purification technology based on molecular structural binding interactions (e.g., ligand-receptor interactions) in which a selective surface-immobilized ligand recognizes and binds to the biological product to be purified. . For example, the first module has at least one first inlet and at least one first outlet, and allows continuous fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick and place robotic system. It consists of

In embodiments, the affinity-based magnetic purification device further comprises a suspension of magnetic resin beads. The surface of the magnetic resin beads is linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer. Continuous purification of biological products (eg, monoclonal antibodies) with affinity magnetic resin beads avoids cumbersome processing steps of conventional affinity column chromatography (eg, Protein A affinity chromatography).

In embodiments, the magnetic resin beads of the affinity-based magnetic purification device have a diameter of about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the magnetic resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of the magnetic resin beads may be about 1% by weight to about 10% by weight. In another example, the binding capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

A loop conveyor system may refer, for example, to a continuous or endless loop. Loop conveyor systems can move large volumes continuously and efficiently at high flow rates through the process while allowing for a smaller footprint compared to conventional affinity column chromatography systems (e.g., Protein A affinity chromatography). advantageous in that Biological products are transported directly on the track, so regular or irregularly shaped objects of any size can be configured for transport. In some aspects, the object is a shipping container having a regular shape (eg, cube, rectangular prism, cylinder, and cone).

In embodiments, a loop conveyor system includes continuously receiving a filtrate comprising a mixture comprising a biological product, followed by the resulting heterogeneous mixture comprising a biological product, magnetic resin beads, a buffer, or any combination thereof. There are at least two transport containers filled with magnetic resin beads configured to transport the

A pick-and-place robotic system may refer to, for example, at least one robot or robotic arm. Pick-and-place robotic systems can move large volumes continuously and efficiently at high flow rates through the process while allowing for a smaller footprint compared to conventional affinity column chromatography systems (e.g., Protein A affinity chromatography). It is advantageous in that there is Since the biological product contained in the transport container is picked up and placed, regular shaped objects of all sizes, with or without handles, can be configured for transport and loading. In some aspects, the object is a shipping container having regular shapes (eg, cubes, and rectangular prisms).

In embodiments, the pick-and-place robotic system includes continuously receiving a filtrate comprising a mixture comprising a biological product, followed by the resulting inhomogeneous material comprising the biological product, magnetic resin beads, buffer, or any combination thereof. There are at least two transport vessels filled with magnetic resin beads configured to transport the mixture.

The affinity-based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable cleaning within at least one of the at least two transport vessels. include additional Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable elution of the biological product within at least one of the at least two transport vessels. Alternatively, the at least one external magnetic field may be used to enable recycling of the magnetic resin beads within at least one of the at least two transport containers. For example, mixing of the magnetic resin beads can be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

The processes described herein also include a biological product from at least one first outlet of the first module into a second module having at least one inlet for receiving flow from the at least one first outlet of the first module. and continuously delivering the fraction to which the second module comprises a charge-based magnetic purification device. As used herein, "charge-based magnetic purification device" includes purifying a biological molecule based, for example, on its surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, charge-based magnetic purification devices include positive charge-based magnetic purification devices, negative charge-based magnetic purification devices, or combinations thereof. For example, the second module has at least one second inlet and at least one second outlet and allows continuous fluid flow between the second inlet and the second outlet via a loop conveyor system or pick and place robotic system. It consists of

In embodiments, the charge-based magnetic purification device (eg, positive charge and/or negative charge-based magnetic purification) further comprises a suspension of magnetic resin beads. For example, the surface of the magnetic resin beads comprises cationic or anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based magnetic purification or negative charge-based magnetic purification, respectively. can do. Continuous purification of biological products (e.g., monoclonal antibodies) with ionic magnetic resin beads avoids cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography). .

In embodiments, the magnetic resin beads of the charge-based magnetic purification device have a diameter of about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the magnetic resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of the magnetic resin beads may be about 1% by weight to about 10% by weight. In another example, the charge or electrostatic association capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

A loop conveyor system may refer, for example, to a continuous or endless loop. Loop conveyor systems are advantageous in that they can move large volumes continuously and efficiently at high flow rates through the process while allowing a smaller footprint compared to conventional ion exchange column chromatography systems. Biological products are transported directly on the track, so regular or irregularly shaped objects of any size can be configured for transport. In some aspects, the object is a shipping container having a regular shape (eg, cube, rectangular prism, cylinder, and cone).

In embodiments, a loop conveyor system is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, buffer, or any combination thereof. There are at least two transport containers filled with magnetic resin beads.

A pick-and-place robotic system may refer to, for example, at least one robot or robotic arm. Pick-and-place robotic systems are advantageous in that they can move large quantities continuously and efficiently at high flow rates through the process while allowing a smaller footprint compared to conventional ion exchange chromatography systems. Since the biological product contained in the transport container is picked up and placed, regular shaped objects of all sizes, with or without handles, can be configured for transport and loading. In some aspects, the object is a shipping container having regular shapes (eg, cubes, and rectangular prisms).

In embodiments, the pick-and-place robotic system includes continuously receiving a filtrate comprising a mixture comprising a biological product, followed by the resulting inhomogeneous material comprising the biological product, magnetic resin beads, buffer, or any combination thereof. There are at least two transport vessels filled with magnetic resin beads configured to transport the mixture.

The charge-based magnetic purification module adds at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable cleaning within at least one of the at least two transport vessels. to include Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable dissociation and collection of the biological product within at least one of the at least two transport vessels. there is. Alternatively, the at least one external magnetic field may be used to enable recycling of the magnetic resin beads within at least one of the at least two transport containers. For example, mixing of the magnetic resin beads can be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

In embodiments described herein, the magnetic resin beads of one or both of the first (affinity-based magnetic purification) and/or second (charge-based magnetic purification) module(s) are recycled and reused. For example, the beads can be reused at least 2, 3, 4, or more times to purify a biological product.

Alternatively, the processes described herein may direct the biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. wherein the second module comprises a free flow electrophoresis device. A free flow electrophoresis device having a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and an aqueous ionic solution is a charge for purifying a biological product (e.g., a monoclonal antibody). based magnetic purification module(s) may be used in place of or in addition to it.

For example, the solution contacting surfaces of the two parallel plates include glass, ceramic, plastic, or any combination thereof. In some instances, an aqueous ionic solution may generate a pH gradient across the main separation channel. In another example, an aqueous ionic solution may impart a constant pH across the main separation channel.

In embodiments, a free flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module may have a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (eg, the approximate pH gradient may range from about 2 to about 10) at least one first fluid element; and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separating channel (e.g., the fine pH gradient is a pH of about 5 to about 8). and at least one second fluid element including a range). For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In another embodiment, a free flow electrophoretic device comprises an electric field or electric field gradient perpendicular to the direction of fluid flow and a fluid channel created between two parallel plates for operating in a zeolite electrophoresis or charge separation mode of operation, wherein the pH There is at least one fluidic element with no gradient (eg, constant pH in the main separation channel). For example, an isoelectric point-based fluid purification module includes a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7). at least one first fluid element; and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (eg, less than pH 7). .

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates to operate in an isokinetic electrophoretic mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a spacer solution (e.g. , NaCl solution) and at least one fluidic element comprising both an acidic pH gradient and a basic pH gradient.

In another embodiment, the isoelectric point-based fluid purification module includes at least one first free flow electrophoresis device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and 2 at least one second free flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, wherein each element is connected in series; can operate in an independent operating mode that enables For example, at least one first free-flow electrophoresis device may operate in an isoelectric focusing mode and at least one second free-flow electrophoresis device may operate in an isokinetic mode to increase separation resolution.

In another embodiment, an isoelectric based fluid purification module includes at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; A fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g., in the pH range of about 2 to about 10). at least one second free flow electrophoresis device comprising; and a fluid channel created between the two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. and at least one third free flow electrophoresis device. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (eg, a platinum wire electrode) to function as an anode or cathode.

In embodiments, backpressure within an isoelectric point-based fluid purification device depends on channel shape and dimensions, inlet and outlet openings and/or tubing diameters, and input flow rate. For example, the back pressure ranges from about 0.5 psi to about 10 psi. In some examples, the back pressure is controlled by, for example, without intending to limit, a needle valve.

In embodiments, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar.

In embodiments, the isoelectric point-based fluid purification device further includes an active cooling system or heat sink (e.g., a Peltier element, a thermal chuck with a circulating water/propylene glycol jacket) to enable temperature control and Joule heat dissipation. do. For example, the active cooling system can control cooling and/or heat dissipation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Ideally, the temperature is maintained between about 10° C. and about 25° C. when isolating biological products (eg, monoclonal antibodies). For example, an active cooling system includes an aluminum thermal chuck with a cooled circulating water/propylene glycol jacket.

In embodiments, an isoelectric point based fluid purification module includes at least one buffer or amphoteric electrolyte system.

In embodiments, an isoelectric point based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper functioning in contact with an anode or cathode, for example phosphoric acid and sodium hydroxide, respectively. In another embodiment, at least one electrode solution is configured to contact the anode or cathode to enable proper function, such as Tris buffered saline, flowing through the main separator channel, the anode channel, and the cathode channel. Contains an amphoteric electrolyte solution.

In embodiments, an isoelectric based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is arranged in-line. In some examples, the at least one sensor or detector includes, but is not limited to, a flow sensor, temperature sensor, conductivity sensor, pH sensor, refractive index detector, UV detector, or back pressure sensor.

In embodiments, an isoelectric point-based fluid purification module includes at least one liquid circuit breaker or disconnects downstream of the device and upstream of at least one in-line sensor or detector to perform sensing or detection in a voltage-free solution. make it possible

The presently claimed process is compatible with current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. It offers many advantages over For example, without intending to be limiting, the processes described herein can reduce production facility footprint, facility build-up, when compared to existing approaches to making batch, single use, or semi-continuous monoclonal antibodies while maintaining throughput and yield. and a continuous bioprocess for purifying monoclonal antibodies that significantly reduces the time required for validation, costs associated with facility construction, and capital equipment expenditure. Continuous bioprocessing as described herein, due to its ability to operate continuously, is the large process equipment required for the batch centrifugation, depth filtration, and column chromatography steps of existing downstream bioprocessing - its size is large. Dependent on bioreactor volume - allows the use of smaller and streamlined equipment (eg, smaller bioreactor volume and downstream bioprocess equipment) as it is not required. Additionally, smaller and streamlined equipment that operates continuously allows the use of much smaller bioreactor(s) that produce monoclonal antibodies at steady state. A continuous bioprocess as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and biological product loss when compared to existing monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems having a footprint that occupies significantly fewer square feet than current technologies without sacrificing product throughput or yield on a kilograms per year basis.

Advantages of the processes and methods described herein include the ability to remove large impurities (eg, cells, cell debris, and aggregates) without membrane fouling or clogging. Membrane fouling can refer to a process in which a heterogeneous mixture is deposited on the membrane surface or within the membrane pores, resulting in a decrease in performance of the membrane over time, greatly limiting the utility of existing filtration systems. For example, clarification of cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or blockage of the filter membrane, making such methods unsuitable for long-term continuous processes. making it unsuitable as a means for the continuous removal of large impurities from heterogeneous mixtures containing biological products through In contrast, the dynamic filtration devices described herein enable continuous removal of large impurities from heterogeneous mixtures containing biological products without fouling the membranes, as the active target area of the filter membrane is continuously renewed.

Additionally, the entire process of producing and purifying the biological product may be continuous, with a flow rate ranging from about 0.1 mL/min to about 50 mL/min (eg, from about 5 mL/min to about 10 mL/min) throughout the process. can be maintained, the process equipment and overall process footprint can occupy a much smaller footprint than current standard processes without sacrificing product throughput or yield on a kilogram/year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require at least 200,000 square feet. For example, the flow rate of a process to purify a biological product ranges from about 1 mL/min to about 10 mL/min. In some instances, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL/min to about 50 mL/min. In another example, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor discharge line. In another example, a process is provided wherein the flow rate of the step of continuously delivering the filtrate to the first module ranges from about 0.1 mL/min to about 50 mL/min. In another example, a process is provided wherein the flow rate of the step of continuously transferring a fraction comprising biological products from the first outlet to the second module ranges from about 0.1 mL/min to about 50 mL/min.

An important advantage of the processes and methods using magnetic resin beads (eg, magnetic agarose) described herein is that such systems can be used in existing stationary phase or packed resin columns (eg, magnetic agarose) for sterilization, recycling, and/or regeneration. , for standard chromatography). For example, such a system provides recycling and/or regeneration of magnetic resin beads to create an infinite surface area of magnetic resin beads during operation, resulting in a continuous and cost effective method. In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, magnetic resin beads used during the purification of a biological product as described herein are continuously recycled and regenerated, such that the flow from the bioreactor discharge line is not interrupted in either the dynamic filtration module or the purification module from the previous step. flow can be accommodated.

In other words, because the modules described in the present invention pass through these steps sequentially, they do not need to be left idle for sterilization, regeneration, and/or recycling after execution. The method requires several packed layers to accommodate a continuous input flow and enable regeneration and/or recycling of columns that have reached full capacity, as current column chromatography methods have limited column capacity limitations due to resin packing constraints. It differs from current continuous chromatography methods in that column switching of columns is required. Another advantage of the method described herein includes that the magnetic resin beads do not pack into the stationary phase, but rather the magnetic resin beads move. The mobility of these beads increases the surface area of the resin beads available for bonding or association since substantially more of the surface of the magnetic resin beads is exposed and free to bond. Additionally, the resin beads within the packed column are exposed to high pressure differentials to create flow through the column, which damage is one of the reasons the column shortens its desired life. The mobile resin beads of the presently described invention are subjected to substantially lower pressures and therefore are much softer to the fragile beads, thereby extending their lifespan. Additionally, this mobility increases the possibility that the magnetic resin beads will complete regeneration and return to their initial state. This further increases the cost effectiveness of the methods described herein because the magnetic resin is utilized more efficiently.

An important advantage of the processes and methods using free flow electrophoresis described herein is that the system exhibits a “product loss free” process, i.e., the separation is dependent on the physicochemical properties of the target biological product in an aqueous solution and is dependent on the electrical field. The point is that the product does not have to interact with the resin or other purifying moiety because it occurs via interaction. Another advantage is observed in the resolution of this approach, as compared to conventional ion exchange chromatography, theoretically a higher purity product can be obtained. In addition, separation based on intrinsic physiochemical properties may be performed on proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentivirus, but extends the usefulness of this approach for the purification of a variety of biological products.

In addition, the modular approach provides flexibility in process design to accommodate a wide range of biological products.

A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based purification module, and a charge-based purification module or an isoelectric point-based fluid purification module.

A continuous process for purifying a biological product is described, said process comprising continuously receiving through an input line a heterogeneous mixture comprising the biological product, wherein the biological product is a protein or fragment thereof (polypeptide), an antibody or fragments thereof, cytokines, chemokines, enzymes, or growth factors. Upon purification, biological products (e.g., monoclonal antibodies) are free from impurities (cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts). , buffer components, surfactants, sugars, metal contaminants, leachates, media components, and/or naturally occurring organic molecules with which they are naturally associated) at least about 60%, 70%, 80%, 90% by weight , 95% by weight, or even 99% by weight is substantially pure.

The process involves the continuous removal of large impurities from the heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module generating a filtrate comprising biological product by continuously supplying biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure. The dynamic filtration module may further include at least one additional input line for supplying wash buffer through a coaxial output head or a separate monoaxial output head.

In embodiments, the processes described herein include purifying biological products produced continuously in bioreactors (eg, fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, a bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, the output bleed line providing continuous fluid flow from the bioreactor to the dynamic filtration module. It serves as an input line allowing flow.

As described herein, processes that continuously remove large impurities from heterogeneous mixtures do not include centrifugation, disk-stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. don't The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, a filter membrane (or depth filter) has a finite capacity and when the membrane reaches its capacity it is filtered. The rate decreases (eg membrane pores become clogged). In “static” (as opposed to “dynamic”) filtration, the filter membrane remains stationary (does not move) and a flow (eg, flow of a heterogeneous mixture) passes through a stationary filter membrane. Such static filtration methods are common in the art, simple and well known.

Unlike static filtration methods commonly used in the art, the process herein describes a dynamic filtration module, wherein the components of a dynamic filtration module allow filtration to continuously occur in new, unused target areas of the filter membrane. (e.g., the membrane moves or advances depending on the flow rate of the overall process). This prevents membrane fouling or clogging and allows control of filter cake packing and thickness during operation.

The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, a vacuum line, a vacuum system, and at least one vacuum collection vessel.

For example, the filter membrane roll includes a rolled filter membrane, wherein the filter membrane is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, poly carbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

The pore size of the rolled filter membrane depends on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

The filter membrane roll has a width of about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system or membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts with a prefabricated roll and continues to an initially empty collection roll to create a reel-to-reel system. In aspects, the dynamic filtration module includes a rolled filter membrane extending between the supply reel and the collection reel, the filter membrane having an active target area configured to receive the heterogeneous mixture. For example, feed reel movement is controlled by a servo motor coupled with a gearbox that limits revolutions per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collecting reel movement is controlled by a servo motor coupled with a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. Additionally, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure consistent speed with the constantly changing diameter of the filter membrane rolls on both the feed and collect reels during operation. For example, the supply reel and the collection reel run in the same direction at the same speed.

In embodiments, the transport rate of the filter membrane ranges from about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1 mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includes a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg PTFE), and an opening continuous with the vacuum line. As used herein, “membrane support structure” refers to a component constructed to provide structural support to the active area of a filter membrane to prevent deformation as it passes through a negative pressure area due to an opening continuous with the vacuum line. Also, as used herein, a “mechanically smooth contact surface” refers to a surface that has a low static coefficient of friction, particularly when wet, that creates a low frictional force opposing the transport of the filter membrane. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, the lower the value the smoother the surface. Also, because rough surfaces have greater friction between surfaces than smooth surfaces, mechanically smooth contact surfaces as used herein refer to surfaces with low friction (ie, low static coefficient of friction).

In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (eg, a mesh, at least one slot, at least one hole, or any combination thereof). The opening may also include regularly spaced elements, for example, the opening may include a series of equally spaced parallel slots. Additionally, the opening may comprise a grate (eg, a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, where each grate is vertical. The opening may be a collection of irregular or regular elements (eg a series of parallel slots). The openings may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (eg, mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without intending to be limiting, the openings may include a mesh between about 0.25 mm and about 5 mm thick.

The membrane support structure of the dynamic filtration module contains a temperature control mechanism. The temperature control mechanism maintains the temperature from about 4° C. to about 37° C. when there is evaporative cooling. For example, during antibody purification, the temperature control mechanism maintains a temperature between about 15°C and about 37°C. Exemplary temperature control mechanisms include, but are not limited to, single loop controllers, multiple loop controllers, closed loop controllers, PID controllers, Peltier elements, and/or thermal chucks with circulating water/propylene glycol jackets.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, PTFE, PFA). For example, the dynamic filtration module includes at least one support rod or roller with a mechanically flat contact surface to stabilize the movement of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to an active target area of the filter membrane. For example, the at least one output head is a tube or slot die.

In some embodiments, the dynamic filtration module includes at least one additional input line for supplying wash buffer through a coaxial output head, a separate monoaxial output head, a separate slot die output head, or a slot die output head with multiple openings. additionally includes

In some embodiments, the dynamic filtration module may include elements known in the coatings and converting industry, such as, but not intended to be limiting, active or passive edge guides, tension regulators (e.g., dancers), brakes and tension detectors; or any combination thereof.

In embodiments, a dynamic filtration module includes a vacuum system in continuity with a membrane support structure to apply a negative pressure over a target area (eg, an active target area) of a filter membrane, wherein the negative pressure is applied to the membrane support structure. It allows active transport of the filter membrane across and allows collection of the filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. In aspects described herein, during purification by dynamic filtration, the filtrate comprising biological products is fed under negative pressure to a vacuum collection vessel capable of collecting about 50 mL to about 100 L. For example, a vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 50 L.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process may include a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced.

The process described herein involves continuously passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product. For example, separating a solution into two or more fractions may include at least one fraction comprising biological products, and at least one other fraction comprising minor impurities. As described herein, the first module includes an affinity-based purification device. "Affinity-based purification device" refers to a purification technology based on molecular structural binding interactions (eg, ligand-receptor interactions) in which a selective surface immobilized ligand recognizes and binds to the biological product to be purified. For example, the first module has at least one first inlet and at least one first outlet, the first inlet and the first outlet via a mechanical rotation system comprising a lid system, a container carousel, and a collection system. configured to allow continuous fluid flow between them.

In embodiments, the affinity-based purification device further comprises a suspension of resin beads. The surface of the resin beads is linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer. Continuous purification of biological products (eg, monoclonal antibodies) with affinity resin beads avoids cumbersome processing steps of conventional affinity column chromatography (eg, Protein A affinity chromatography).

In embodiments, the diameter of the resin beads of the affinity-based purification device is between about 0.2 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of the magnetic resin beads may be about 1% by weight to about 20% by weight. In another example, the binding capacity of the resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, an affinity-based purification module includes a lid system having at least one gasket lid, the at least one gasket lid comprising: at least one inlet for introducing a gas to control a static head pressure; at least one vent port to equalize atmospheric pressure; at least one inlet for introducing a suspension of resin beads; at least one inlet for receiving a filtrate containing biological products; and at least two inlets for introducing a buffer system dispersing the resin beads to enable washing, elution from, or regeneration of the resin beads. In some embodiments, the at least one gasket lid further includes a port for receiving an overhead agitation impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

In embodiments, an affinity-based purification module continuously receives a mixture comprising a mechanical rotational system, eg, a biological product, followed by the biological product, resin beads, buffer, or any combination thereof. and a carousel comprising at least two containers filled with resin beads configured to transport the resulting heterogeneous mixture. For example, the mechanical rotation system is configured to mate with a lid system to enable pressurization. In another example, a mechanical rotation system controls movement or rotation in the xy plane.

In embodiments, each of the at least two vessels of the affinity-based purification module has a supported bottom filter or filter membrane that enables retention of resin beads during the binding, debinding, washing, elution, and regeneration process steps. . For example, at least two vessels further include a valve for controlling liquid flow.

In embodiments, an affinity-based purification module is a collection system capable of interfacing with at least one of the at least two vessels of the mechanical rotation system to collect waste, a fraction comprising a biological product, or any combination thereof. includes For example, the collection system controls movement along the z-axis.

The processes described herein also include a biological product from at least one first outlet of the first module into a second module having at least one inlet for receiving flow from the at least one first outlet of the first module. and continuously delivering the fractions to be purified, wherein the second module includes a charge-based purification device. As used herein, “charge-based purification device” includes purifying a biological molecule based, for example, on its surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, charge-based purification includes positive charge-based purification devices, negative charge-based purification devices, or combinations thereof. For example, the second module has at least one second inlet and at least one second outlet, the second inlet and the second outlet via a mechanical rotation system comprising a lid system, a container carousel, and a collecting system. configured to allow continuous fluid flow between them.

In embodiments, the charge based purification device (eg, positive charge and/or negative charge based purification) further comprises a suspension of resin beads. For example, the surface of the resin beads may include cationic or anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based purification or negative charge-based purification, respectively. Continuous purification of biological products (eg, monoclonal antibodies) with ionic resin beads avoids cumbersome processing steps of conventional ion exchange column chromatography (eg, cation exchange or anion exchange chromatography).

In embodiments, the diameter of the resin beads of the charge based purification device is between about 0.2 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of the magnetic resin beads may be about 1% by weight to about 20% by weight. In another example, the charge or electrostatic association capacity of resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, a charge-based purification module includes a lid system having at least one gasket lid, the at least one gasket lid comprising: at least one inlet for gas introduction to enable control of a static head pressure; at least one vent port to equalize atmospheric pressure; at least one inlet for introducing a suspension of resin beads; at least one inlet for receiving a filtrate comprising biological products; and at least two inlets for introducing a buffer system dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. In some embodiments, the at least one gasket lid further includes a port for receiving an overhead agitation impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

In embodiments, the charge-based purification module continuously receives a mixture comprising a mechanical rotating system, eg, a biological product, followed by production comprising the biological product, resin beads, buffer, or any combination thereof. and a carousel including at least two containers filled with resin beads configured to transport the heterogeneous mixture. For example, the mechanical rotation system is configured to mate with a lid system to enable pressurization. In another example, a mechanical rotation system controls movement or rotation in the xy plane.

In embodiments, each of the at least two vessels of the charge-based purification module has a supported bottom filter or filter membrane that enables retention of resin beads during association, dissociation, washing, and regeneration process steps. For example, at least two vessels further include a valve for controlling liquid flow.

In embodiments, the charge-based purification module comprises a collection system capable of interfacing with at least one of the at least two vessels of the mechanical rotation system to collect waste, a fraction comprising a biological product, or any combination thereof. include For example, the collection system controls movement along the z-axis.

In embodiments described herein, the resin beads of one or both of the first (affinity-based purification) and/or second (charge-based purification) module(s) are recycled and reused. For example, the beads may be reused at least 2, 3, 4, or more times to purify a biological product.

Alternatively, the processes described herein may direct the biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. wherein the second module comprises a free flow electrophoresis device. A free flow electrophoretic device having a fluidic channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous ionic solution provides a charge for purifying a biological product (e.g., a monoclonal antibody). based magnetic purification module(s) may be used in place of or in addition to it.

For example, the solution contacting surfaces of the two parallel plates include glass, ceramic, plastic, or any combination thereof. In some instances, an aqueous ionic solution may generate a pH gradient across the main separation channel. In another example, an aqueous ionic solution may impart a constant pH across the main separation channels.

In embodiments, a free flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module may have a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (eg, the approximate pH gradient may range from about 2 to about 10) at least one first fluid element; and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separating channel (e.g., the fine pH gradient is a pH of about 5 to about 8). and at least one second fluid element including a range). For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates for operating in a bandelectrophoresis or charge separation mode of operation and an electric field or electric field gradient perpendicular to the direction of fluid flow, wherein the pH There is at least one fluid element with no gradient. For example, an isoelectric point-based fluid purification module includes a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7). at least one first fluid element; and at least one second fluid element comprising a fluid channel created between the two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (eg, less than pH 7). .

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates to operate in an isokinetic electrophoretic mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a spacer solution (e.g. , NaCl solution) and at least one fluidic element comprising both an acidic pH gradient and a basic pH gradient.

In another embodiment, the isoelectric point-based fluid purification module includes at least one first free flow electrophoresis device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and 2 at least one second free flow electrophoresis device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, wherein each element is connected in series; can operate in an independent operating mode that enables For example, at least one first free-flow electrophoresis device may operate in an isoelectric focusing mode and at least one second free-flow electrophoresis device may operate in an isokinetic mode to increase separation resolution.

In another embodiment, an isoelectric based fluid purification module includes at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; A fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g., in the pH range of about 2 to about 10). at least one second free flow electrophoresis device; and a fluid channel created between the two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. and at least one third free flow electrophoresis device. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (eg, a platinum wire electrode) to function as an anode or cathode.

In embodiments, backpressure within an isoelectric point-based fluid purification device depends on channel shape and dimensions, inlet and outlet openings and/or tubing diameters, and input flow rate. For example, the back pressure ranges from about 0.5 psi to about 10 psi. In some examples, the back pressure is controlled by, for example, without intending to limit, a needle valve.

In embodiments, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar.

In embodiments, the isoelectric point-based fluid purification device further includes an active cooling system or heat sink (e.g., a Peltier element, a thermal chuck with a circulating water/propylene glycol jacket) to enable temperature control and Joule heat dissipation. do. For example, the active cooling system can control cooling and/or heat dissipation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Ideally, the temperature is maintained between about 10° C. and about 25° C. when isolating biological products (eg, monoclonal antibodies). For example, an active cooling system includes an aluminum thermal chuck with a cooled circulating water/propylene glycol jacket.

In embodiments, an isoelectric point based fluid purification module includes at least one buffer or amphoteric electrolyte system.

In embodiments, an isoelectric point based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper functioning in contact with an anode or cathode, for example phosphoric acid and sodium hydroxide, respectively. In another embodiment, at least one electrode solution is configured to contact the anode or cathode to enable proper function, such as Tris buffered saline, flowing through the main separator channel, the anode channel, and the cathode channel. Contains an amphoteric electrolyte solution.

In embodiments, an isoelectric based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is arranged in-line. In some examples, the at least one sensor or detector includes, but is not limited to, a flow sensor, temperature sensor, conductivity sensor, pH sensor, refractive index detector, UV detector, or back pressure sensor.

In embodiments, an isoelectric point-based fluid purification module includes at least one liquid circuit breaker or disconnects downstream of the device and upstream of at least one in-line sensor or detector to perform sensing or detection in a voltage-free solution. make it possible

The presently claimed process is compatible with current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. It offers many advantages over For example, without intending to be limiting, the processes described herein can reduce the production facility footprint, facility build-up, when compared to existing approaches to making batch, single use, or semi-continuous monoclonal antibodies while maintaining throughput and yield. and a continuous bioprocess for purifying monoclonal antibodies that significantly reduces the time required for validation, costs associated with facility construction, and capital equipment expenditure. Continuous bioprocessing as described herein, due to its ability to operate continuously, requires large process equipment - the size of which is a bioreactor - required for the centrifugation, depth filtration, and column chromatography steps of existing downstream bioprocessing. volume dependent - allows for the use of smaller and streamlined equipment (eg, smaller bioreactor volume and downstream bioprocess equipment) since no volume dependent is required. Additionally, smaller and streamlined equipment that operates continuously allows the use of much smaller bioreactor(s) that produce monoclonal antibodies at steady state. A continuous bioprocess as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and biological product loss when compared to existing monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems having a footprint that occupies significantly fewer square feet than current technologies without sacrificing product throughput or yield on a kilograms per year basis.

Advantages of the processes and methods described herein include the ability to remove large impurities (eg, cells, cell debris, and aggregates) without membrane fouling or clogging. For example, clarification of cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or blockage of the filter membrane, making such methods unsuitable for long-term continuous processes. making it unsuitable as a means for the continuous removal of large impurities from heterogeneous mixtures containing biological products through In contrast, the dynamic filtration devices described herein enable continuous removal of large impurities from heterogeneous mixtures containing biological products without fouling the membranes, as the active target area of the filter membrane is continuously renewed. Additionally, since the entire process of producing and purifying the biological product can be continuous and flow rates ranging from about 0.1 mL/min to about 50 mL/min can be maintained throughout the process, the process equipment and overall process footprint can be in the order of kilograms. It can occupy a much smaller footprint than current standard processes without sacrificing product throughput or yield on a per-year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require a minimum of 200,000 square feet. For example, the flow rate of a process for purifying a biological product ranges from about 1 mL/min to about 10 mL/min. In some instances, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL/min to about 50 mL/min. In another example, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor discharge line. In another example, a process is provided wherein the flow rate of the step of continuously delivering the filtrate to the first module ranges from about 0.1 mL/min to about 50 mL/min. In another example, a process is provided wherein the flow rate of the step of continuously transferring a fraction comprising biological products from the first outlet to the second module ranges from about 0.1 mL/min to about 50 mL/min.

An important advantage of the processes and methods using resin beads (eg, agarose) described herein is that these systems can be used with existing stationary phase or packed resin columns (eg, standard for chromatography) is not required. For example, such a system provides for recycling and/or regeneration of resin beads to create an infinite surface area of resin beads during operation, resulting in a continuous and cost effective method. In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, the resin beads used during the purification of biological products as described herein are continuously recycled and regenerated so that the flow from the previous step in either the dynamic filtration module or the purification module is not stopped without interrupting the flow from the bioreactor discharge line. can accommodate In other words, because the modules described in the present invention pass through these steps sequentially, they do not need to be left idle for sterilization, regeneration, and/or recycling after execution. The method requires several packed layers to accommodate continuous input flow and to allow regeneration and/or recycling of columns that have reached full capacity, as current column chromatography methods have finite column capacity limitations due to column packing constraints. It differs from current continuous chromatography methods in that column switching of columns is required. Another advantage of the methods described herein includes that the resin beads do not pack in the stationary phase, but rather the resin beads move. The mobility of these beads increases the surface area of the resin beads available for bonding or association since substantially more of the resin bead surface is exposed and free to bond. Additionally, the resin beads within the packed column are exposed to high pressure differentials to create flow through the column, which damage is one of the reasons the column shortens its desired life. The mobile resin beads of the presently described invention are subjected to substantially lower pressures and thus are much softer to the fragile beads, thereby extending their lifespan. Additionally, this mobility increases the possibility that the resin beads will complete regeneration and return to their initial state. This also makes the method described herein more cost effective because the resin is used more efficiently.

An important advantage of the processes and methods using free flow electrophoresis described herein is that the system exhibits a “product loss free” process, i.e., the separation is dependent on the physicochemical properties of the target biological product in an aqueous solution and is dependent on the electrical field. The point is that the product does not have to interact with the resin or other purifying moiety because it occurs via interaction. Another advantage is observed in the resolution of this approach, as compared to conventional ion exchange chromatography, theoretically a higher purity product can be obtained. In addition, separation based on intrinsic physiochemical properties may be performed on proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentivirus, but extends the usefulness of this approach for the purification of a variety of biological products.

In addition, the modular approach provides flexibility in process design to accommodate a wide range of biological products.

A continuous process of purifying a biological product using at least one of a dynamic filtration module, an affinity-based fluid purification module, and a charge-based fluid purification module or an isoelectric point-based fluid purification module.

A continuous process for purifying a biological product is described, said process comprising continuously receiving through an input line a heterogeneous mixture comprising the biological product, wherein the biological product is a protein or fragment thereof (polypeptide), an antibody or fragments thereof, cytokines, chemokines, enzymes, or growth factors. Upon purification, biological products (e.g., monoclonal antibodies) are free from impurities (e.g., cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids, and oligonucleotides). , viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, media components, and/or naturally occurring organic molecules with which they are naturally associated) at least about 60%, 70%, 80% by weight , is substantially pure when 90%, 95%, or even 99% by weight is removed.

The process involves the continuous removal of large impurities from the heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module generating a filtrate comprising biological product by continuously supplying biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure. The dynamic filtration module may further include at least one additional input line for supplying wash buffer through a coaxial output head or a separate monoaxial output head.

In embodiments, the processes described herein include purifying biological products produced continuously in bioreactors (eg, fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, a bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, the output bleed line providing continuous fluid flow from the bioreactor to the dynamic filtration module. It serves as an input line allowing flow.

As described herein, processes that continuously remove large impurities from heterogeneous mixtures do not include centrifugation, disk-stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. don't The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, a filter membrane (or depth filter) has a finite capacity and when the membrane reaches its capacity it is filtered. The rate decreases (eg membrane pores become clogged). In “static” (as opposed to “dynamic”) filtration, the filter membrane remains stationary (does not move) and a flow (eg, flow of a heterogeneous mixture) passes through a stationary filter membrane. Such static filtration methods are common in the art, simple and well known.

Unlike static filtration methods commonly used in the art, the process herein describes a dynamic filtration module, wherein the components of a dynamic filtration module allow filtration to continuously occur in new, unused target areas of the filter membrane. (e.g., the membrane moves or advances depending on the flow rate of the entire process). This prevents membrane fouling or clogging and allows control of filter cake packing and thickness during operation.

The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, a vacuum line, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll comprises a filter roll, wherein the filter membrane is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

The pore size of the rolled filter membrane depends on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

The filter membrane roll has a width of about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system or membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts with a prefabricated roll and continues to an initially empty collection roll to create a reel-to-reel system. In aspects, the dynamic filtration module includes a rolled filter membrane extending between the supply reel and the collection reel, the filter membrane having an active target area configured to receive the heterogeneous mixture. For example, feed reel movement is controlled by a servo motor coupled with a gearbox that limits revolutions per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collecting reel movement is controlled by a servo motor coupled with a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. Additionally, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure consistent speed with the constantly changing diameter of the filter membrane rolls on both the feed and collect reels during operation. For example, the supply reel and the collection reel run in the same direction at the same speed.

In embodiments, the transport rate of the filter membrane ranges from about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1 mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includes a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg PTFE), and an opening continuous with the vacuum line. As used herein, “membrane support structure” refers to a component constructed to provide structural support to the active area of a filter membrane to prevent deformation as it passes through a negative pressure area due to an opening continuous with the vacuum line. Also, as used herein, a “mechanically smooth contact surface” refers to a surface that has a low static coefficient of friction, particularly when wet, that creates a low frictional force opposing the transport of the filter membrane. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, the lower the value the smoother the surface. Also, because rough surfaces have greater friction between surfaces than smooth surfaces, mechanically smooth contact surfaces as used herein refer to surfaces with low friction (ie, low static coefficient of friction).

In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (eg, a mesh, at least one slot, at least one hole, or any combination thereof). The opening may also include regularly spaced elements, for example, the opening may include a series of equally spaced parallel slots. Additionally, the opening may comprise a grate (eg, a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, where each grate is vertical. The opening may be a collection of irregular or regular elements (eg a series of parallel slots). The openings may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (eg, mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without intending to be limiting, the openings may include a mesh between about 0.25 mm and about 5 mm thick.

The membrane support structure of the dynamic filtration module contains a temperature control mechanism. The temperature control mechanism maintains the temperature from about 4° C. to about 37° C. when there is evaporative cooling. For example, during antibody purification, a temperature control mechanism maintains a temperature between 15°C and 37°C. Exemplary temperature control mechanisms include, but are not limited to, single loop controllers, multiple loop controllers, closed loop controllers, PID controllers, Peltier elements, resistive heating elements, and/or thermal chucks with circulating water jackets.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, PTFE, PFA). For example, the dynamic filtration module includes at least one support rod or roller with a mechanically flat contact surface to stabilize the movement of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to a target area (eg, an active target area) of the filter membrane. For example, the at least one output head is a tube or slot die.

In some embodiments, the dynamic filtration module includes at least one additional input line for supplying wash buffer through a coaxial output head, a separate monoaxial output head, a separate slot die output head, or a slot die output head with multiple openings. additionally includes

In some embodiments, the dynamic filtration module may include elements known in the coatings and converting industry, such as, but not intended to be limiting, active or passive edge guides, tension regulators (eg, dancers), brakes and tension detectors; or any combination thereof.

In embodiments, a dynamic filtration module includes a vacuum system in continuity with a membrane support structure to apply a negative pressure across an active target area of a filter membrane, wherein the negative pressure causes active transport of the filter membrane across the membrane support structure. and allow collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. In aspects described herein, during purification by dynamic filtration, the filtrate comprising biological products is fed under negative pressure to a vacuum collection vessel capable of collecting about 50 mL to about 100 L. For example, a vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 50 L.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process may include a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced.

The process described herein involves continuously passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product. For example, separating a solution into two or more fractions may include at least one fraction comprising biological products, and at least one other fraction comprising minor impurities. As described herein, the first module includes an affinity-based fluid purification device. An "affinity-based fluid purification device" is one in which an optional surface immobilized ligand recognizes and binds to a biological product to be purified with at least one hybrid fluidic device or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic). refers to a purification technology based on exploiting molecular structural binding interactions (eg, ligand-receptor interactions) to For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet.

In embodiments, the affinity-based fluid purification device further comprises a suspension of magnetic resin beads. The surfaces of the magnetic resin beads are linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer. Continuous purification of biological products (eg, monoclonal antibodies) with affinity magnetic resin beads avoids cumbersome processing steps of conventional affinity column chromatography (eg, Protein A affinity chromatography).

In embodiments, the diameter of the magnetic resin beads of the affinity-based fluid purification device is between about 0.2 microns and about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the magnetic resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of magnetic resin beads may be about 1% by weight. In another example, the binding capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

The at least one hybrid fluidic element or chip of the affinity-based fluid purification device may refer to, for example, a microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any combination thereof. In some examples, the fluidic device or chip is a hybrid microfluidic device or chip, eg, a microfluidic device that combines the functionality of cross-flow fluid dynamics with magnetophoretic and dielectrophoretic capabilities, wherein the cross-flow fluid dynamics is controlled by a microchannel design, the magnetophoresis is performed through an external magnetic field, and the dielectrophoresis is performed through a dielectrophoretic electrode. In aspects, a combination of a dielectrophoretic electrode and an external magnetic field is used to manipulate the flow path of magnetic resin beads in a cross-flow microchannel to enable efficient purification at high flow rates (e.g., 0.5 mL/min or greater); , which is a currently unrealized phenomenon in the field of microfluidic dynamics where flow rates are typically limited to μL/hr or μL/min. In another example, a hybrid fluid is a microfluidic device that combines the functionality of cross-flow fluid dynamics with magnetophortic and acoustophoretic capabilities, wherein the cross-flow fluid dynamics are controlled by a microchannel design and the magnetophoretic is externally It is carried out through a magnetic field, and the phonophoresis is carried out through a piezoelectric transducer or crystal. In aspects, a combination of a piezoelectric transducer and an external magnetic field is used to manipulate the flow path of magnetic resin beads in a cross-flow microchannel to enable efficient purification at high flow rates (e.g., 0.5 mL/min or greater); This is a currently unrealized phenomenon in the field of microfluidic dynamics where flow rates are typically limited to μL/hr or μL/min.

In embodiments, the affinity-based fluid purification module further comprises at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange a fraction comprising a biological product.

The processes described herein also include a biological product from at least one first outlet of the first module into a second module having at least one inlet for receiving flow from the at least one first outlet of the first module. wherein the second module comprises a charge-based fluid purification device. As used herein, a "charge-based fluid purification device" means, for example, at least one hybrid fluidic device or chip (e.g., a microfluidic, mesofluidic, millifluidic, macrofluidic device or chip, or any of these any combination of) to purify a biological molecule based on its surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, charge-based fluid purification devices include positive charge-based fluid purification devices, negative charge-based fluid purification devices, or combinations thereof. For example, the second module has at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet.

In embodiments, the charge-based fluid purification device further comprises a suspension of magnetic resin beads. For example, the surface of the magnetic resin beads comprises cationic or anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based fluid purification or negative charge-based fluid purification, respectively. can do. Continuous purification of biological products (e.g., monoclonal antibodies) with ionic magnetic resin beads avoids cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography). .

In embodiments, the diameter of the magnetic resin beads of the charge-based fluid purification device is between about 0.2 microns and about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the magnetic resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of magnetic resin beads may be about 1% by weight. In another example, the charge or electrostatic association capacity of magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

The at least one hybrid fluidic element or chip of the charge-based fluid purification device may refer to, for example, a microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any combination thereof. In some examples, the fluidic device or chip is a hybrid microfluidic device or chip, eg, a microfluidic device that combines the functionality of cross-flow fluid dynamics with magnetophoretic and dielectrophoretic capabilities, wherein the cross-flow fluid dynamics is controlled by a microchannel design, the magnetophoresis is performed through an external magnetic field, and the dielectrophoresis is performed through a dielectrophoretic electrode. In aspects, a combination of a dielectrophoretic electrode and an external magnetic field is used to manipulate the flow path of magnetic resin beads in a cross-flow microchannel to enable efficient purification at high flow rates (e.g., 0.5 mL/min or greater); , which is a currently unrealized phenomenon in the field of microfluidic dynamics where flow rates are typically limited to μL/hr or μL/min. In another example, a hybrid fluid is a microfluidic device that combines the functionality of cross-flow fluid dynamics with magnetophortic and acoustophoretic capabilities, wherein the cross-flow fluid dynamics are controlled by a microchannel design and the magnetophoretic is externally It is carried out through a magnetic field, and the phonophoresis is carried out through a piezoelectric transducer or crystal. In aspects, a combination of a piezoelectric transducer and an external magnetic field is used to manipulate the flow path of magnetic resin beads in a cross-flow microchannel to enable efficient purification at high flow rates (e.g., 0.5 mL/min or greater); This is a currently unrealized phenomenon in the field of microfluidic dynamics where flow rates are typically limited to μL/hr or μL/min.

In embodiments, the charge-based fluid purification module further comprises at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange the fraction comprising the biological product.

In embodiments described herein, the magnetic resin beads of one or both of the first (affinity-based fluid purification) and/or second (charge-based fluid purification) modules are recycled and reused. For example, the beads can be reused at least 2, 3, 4, or more times to purify a biological product.

Alternatively, the processes described herein may direct the biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. wherein the second module comprises a free flow electrophoresis device. A free flow electrophoretic device having a fluidic channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous ionic solution provides a charge for purifying a biological product (e.g., a monoclonal antibody). based magnetic purification module(s) may be used in place of or in addition to it.

For example, the solution contacting surfaces of the two parallel plates include glass, ceramic, plastic, or any combination thereof. In some instances, an aqueous ionic solution may generate a pH gradient across the main separation channel. In another example, an aqueous ionic solution may impart a constant pH across the main separation channel.

In embodiments, a free flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module may have a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (eg, the approximate pH gradient may range from about 2 to about 10); and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separating channel (e.g., the fine pH gradient is a pH of about 5 to about 8). and at least one second fluid element including a range). For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates for operating in a bandelectrophoresis or charge separation mode of operation and an electric field or electric field gradient perpendicular to the direction of fluid flow, wherein the pH There is at least one fluid element with no gradient. For example, an isoelectric point-based fluid purification module includes a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7). at least one first fluid element; and at least one second fluid element comprising a fluid channel created between the two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (eg, less than pH 7). .

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates to operate in an isokinetic electrophoretic mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a spacer solution (e.g. , NaCl solution) and at least one fluidic element comprising both an acidic pH gradient and a basic pH gradient.

In another embodiment, the isoelectric point-based fluid purification module includes at least one first free flow electrophoresis device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and 2 at least one second free flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, wherein each element is connected in series; can operate in an independent operating mode that enables For example, at least one first free-flow electrophoresis device may operate in an isoelectric focusing mode and at least one second free-flow electrophoresis device may operate in an isokinetic mode to increase separation resolution.

In another embodiment, an isoelectric based fluid purification module includes at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; A fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g., in the pH range of about 2 to about 10). at least one second free flow electrophoresis device comprising; and a fluid channel created between the two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. and at least one third free flow electrophoresis device. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (eg, a platinum wire electrode) to function as an anode or cathode.

In embodiments, back pressure within an isoelectric point-based fluid purification device depends on channel shape and dimensions, inlet and outlet openings and/or tubing diameters, and input flow rate. For example, the back pressure ranges from about 0.5 psi to about 10 psi. In some examples, the back pressure is controlled by, for example, without intending to limit, a needle valve.

In embodiments, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar.

In embodiments, the isoelectric point-based fluid purification device further includes an active cooling system or heat sink (e.g., a Peltier element, a thermal chuck with a circulating water/propylene glycol jacket) to enable temperature control and Joule heat dissipation. do. For example, the active cooling system can control cooling and/or heat dissipation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Ideally, the temperature is maintained between about 10° C. and about 25° C. when isolating biological products (eg, monoclonal antibodies). For example, an active cooling system includes an aluminum thermal chuck with a cooled circulating water/propylene glycol jacket.

In embodiments, an isoelectric point based fluid purification module includes at least one buffer or amphoteric electrolyte system.

In embodiments, an isoelectric point based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper functioning in contact with an anode or cathode, for example phosphoric acid and sodium hydroxide, respectively. In other embodiments, at least one electrode solution is configured to contact the anode or cathode to enable proper function, such as Tris buffered saline, flowing through the main separator channel, the anode channel, and the cathode channel. Contains an amphoteric electrolyte solution.

In embodiments, an isoelectric based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is arranged in-line. In some examples, the at least one sensor or detector includes, but is not limited to, a flow sensor, temperature sensor, conductivity sensor, pH sensor, refractive index detector, UV detector, or back pressure sensor.

In embodiments, an isoelectric point-based fluid purification module includes at least one liquid circuit breaker or disconnects downstream of the device and upstream of at least one in-line sensor or detector to perform sensing or detection in a voltage-free solution. make it possible

The presently claimed process is compatible with current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. It offers many advantages over For example, without intending to be limiting, the processes described herein can reduce production facility footprint, facility build-up, when compared to existing approaches to making batch, single use, or semi-continuous monoclonal antibodies while maintaining throughput and yield. and a continuous bioprocess for purifying monoclonal antibodies that significantly reduces the time required for validation, costs associated with facility construction, and capital equipment expenditure. Continuous bioprocessing as described herein, due to its ability to operate continuously, requires large process equipment - the size of which is large bioreactors - for the centrifugation, depth filtration, and column chromatography steps of existing downstream bioprocessing. Volume dependent - allows for the use of smaller and streamlined equipment (eg, smaller bioreactor volume and downstream bioprocess equipment) since no volume dependent is required. Additionally, smaller and streamlined equipment that operates continuously allows the use of much smaller bioreactor(s) that produce monoclonal antibodies at steady state. A continuous bioprocess as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and biological product loss when compared to existing monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems having a footprint that occupies significantly fewer square feet than current technologies without sacrificing product throughput or yield on a kilograms per year basis.

Advantages of the processes and methods described herein include the ability to remove large impurities (eg, cells, cell debris, and aggregates) without membrane fouling or clogging. For example, clarification of cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or blockage of the filter membrane, making such methods unsuitable for long-term continuous processes. making it unsuitable as a means for the continuous removal of large impurities from heterogeneous mixtures containing biological products through In contrast, the dynamic filtration devices described herein enable continuous removal of large impurities from heterogeneous mixtures containing biological products without fouling the membranes, as the active target area of the filter membrane is continuously renewed. Additionally, since the entire process of producing and purifying the biological product can be continuous and flow rates ranging from about 0.1 mL/min to about 50 mL/min can be maintained throughout the process, the process equipment and overall process footprint can be in the order of kilograms. It can occupy a much smaller footprint than current standard processes without sacrificing product throughput or yield on a per-year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require a minimum of 200,000 square feet. For example, the flow rate of a process for purifying a biological product ranges from about 1 mL/min to about 10 mL/min. In some instances, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL/min to about 50 mL/min. In another example, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor discharge line. In another example, a process is provided wherein the flow rate of the step of continuously delivering the filtrate to the first module ranges from about 0.1 mL/min to about 50 mL/min. In another example, a process is provided wherein the flow rate of the step of continuously transferring a fraction comprising biological products from the first outlet to the second module ranges from about 0.1 mL/min to about 50 mL/min. Additionally, the ability for processes to purify biological products at flow rates ranging from about 0.1 mL/min to about 50 mL/min using hybrid microfluidic devices is due to microfluidic dynamics where flow rates are typically limited to μL/hr or μL/min. This is a phenomenon that is currently unrealized in the field.

An important advantage of the processes and methods using magnetic resin beads (eg, magnetic agarose) described herein is that such systems can be used in existing stationary phase or packed resin columns (eg, magnetic agarose) for sterilization, recycling, and/or regeneration. , for standard chromatography). For example, such a system provides recycling and/or regeneration of magnetic resin beads to create an infinite surface area of magnetic resin beads during operation, resulting in a continuous and cost effective method. In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, the magnetic resin beads used during the purification of biological products as described herein are continuously recycled and regenerated, so that the flow from the bioreactor discharge line is not interrupted in either the dynamic filtration module or the purification module from the previous step. flow can be accommodated. In other words, because the modules described in the present invention pass through these steps sequentially, they do not need to be left idle for sterilization, regeneration, and/or recycling after execution. The method requires several packed layers to accommodate a continuous input flow and enable regeneration and/or recycling of columns that have reached full capacity, as current column chromatography methods have limited column capacity limitations due to resin packing constraints. It differs from current continuous chromatography methods in that column switching of columns is required. Another advantage of the method described herein includes that the magnetic resin beads do not pack into a stationary phase, but rather the magnetic resin beads move. The mobility of these beads increases the surface area of the magnetic resin beads available for bonding or association since substantially more of the magnetic resin bead surface is exposed and free to bond. Additionally, the resin beads within the packed column are exposed to high pressure differentials to create flow through the column, which damage is one of the reasons the column shortens its desired life. The mobile resin beads of the presently described invention are subjected to substantially lower pressures and thus are much softer to the fragile beads, thereby extending their lifespan. In addition, this mobility increases the possibility that the magnetic resin beads will complete regeneration and return to their initial state. This also makes the method described herein more cost effective because the resin is used more efficiently.

An important advantage of the processes and methods using free flow electrophoresis described herein is that the system represents a "product loss free" process, i.e., the separation is dependent on the physicochemical properties of the target biological product in an aqueous solution and is dependent on the electrical field. The point is that the product does not have to interact with the resin or other purifying moiety because it occurs via interaction. Another advantage is observed in the resolution of this approach, as compared to conventional ion exchange chromatography, theoretically a higher purity product can be obtained. In addition, separation based on essential physiochemical properties can be performed on proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentivirus, but extends the usefulness of this approach for the purification of a variety of biological products, including but not limited to.

In addition, the modular approach provides flexibility in process design to accommodate a wide range of biological products.

A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based TFF purification module, and a charge-based TFF purification module or an isoelectric point-based fluid purification module.

A continuous process for purifying a biological product is described, said process comprising continuously receiving through an input line a heterogeneous mixture comprising the biological product, wherein the biological product is a protein or fragment thereof (polypeptide), an antibody or fragments thereof, cytokines, chemokines, enzymes, or growth factors. Upon purification, biological products (e.g., monoclonal antibodies) are free from impurities (e.g., cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids, and oligonucleotides). , viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, media components, and/or naturally occurring organic molecules with which they are naturally associated) at least 60%, 70%, 80%, It is substantially pure when 90%, 95%, or even 99% by weight is removed.

The process involves the continuous removal of large impurities from the heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module generating a filtrate comprising biological product by continuously supplying biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure. The dynamic filtration module may further include at least one additional input line for supplying wash buffer through a coaxial output head or a separate monoaxial output head.

In embodiments, the processes described herein include purifying biological products produced continuously in bioreactors (eg, fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, a bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, the output bleed line providing continuous fluid flow from the bioreactor to the dynamic filtration module. It serves as an input line allowing flow.

As described herein, processes that continuously remove large impurities from heterogeneous mixtures do not include centrifugation, disk-stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. don't The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, a filter membrane (or depth filter) has a finite capacity and when the membrane reaches its capacity, it is filtered. The rate decreases (eg membrane pores become clogged). In "static" (as opposed to "dynamic") filtration, the filter membrane remains stationary (does not move) and a flow (eg, flow of a heterogeneous mixture) passes through a stationary filter membrane. Such static filtration methods are common in the art, simple and well known.

Unlike the static filtration methods commonly used in the art, the process herein describes a dynamic filtration module, wherein the components of a dynamic filtration module allow filtration to continuously occur in new, unused target areas of the filter membrane. (e.g., the membrane moves or advances depending on the flow rate of the entire process). This prevents membrane fouling or clogging and allows control of filter cake packing and thickness during operation.

The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, a vacuum line, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll comprises a filter roll, wherein the filter membrane is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

The pore size of the rolled filter membrane depends on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

The filter membrane roll has a width of about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system or membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts with a prefabricated roll and continues to an initially empty collection roll to create a reel-to-reel system. In aspects, the dynamic filtration module includes a rolled filter membrane extending between the supply reel and the collection reel, the filter membrane having an active target area configured to receive the heterogeneous mixture. For example, feed reel movement is controlled by a servo motor coupled with a gearbox that limits revolutions per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collecting reel movement is controlled by a servo motor coupled with a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. Additionally, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure consistent speed with the constantly changing diameter of the filter membrane rolls on both the feed and collect reels during operation. For example, the supply reel and the collection reel run in the same direction at the same speed.

In embodiments, the transport rate of the filter membrane ranges from about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1 mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includes a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg PTFE), and an opening continuous with the vacuum line. As used herein, “membrane support structure” refers to a component constructed to provide structural support to the active area of a filter membrane to prevent deformation as it passes through a negative pressure area due to an opening continuous with the vacuum line. Also, as used herein, a “mechanically smooth contact surface” refers to a surface that has a low static coefficient of friction, particularly when wet, that creates a low frictional force opposing the transport of the filter membrane. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, the lower the value the smoother the surface. Also, because rough surfaces have greater friction between surfaces than smooth surfaces, mechanically smooth contact surfaces as used herein refer to surfaces with low friction (ie, low static coefficient of friction).

In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (eg, a mesh, at least one slot, at least one hole, or any combination thereof). The opening may also include regularly spaced elements, for example, the opening may include a series of equally spaced parallel slots. Additionally, the opening may comprise a grate (eg, a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, where each grate is vertical. The opening may be a collection of irregular or regular elements (eg a series of parallel slots). The openings may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (eg, mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without intending to be limiting, the openings may include a mesh between about 0.25 mm and about 5 mm thick.

The membrane support structure of the dynamic filtration module contains a temperature control mechanism. The temperature control mechanism maintains a temperature between 4°C and 37°C with evaporative cooling. For example, during antibody purification, a temperature control mechanism maintains a temperature between 15°C and 37°C. Exemplary temperature control mechanisms include, but are not limited to, single loop controllers, multiple loop controllers, closed loop controllers, PID controllers, Peltier elements, resistive heating elements, and/or thermal chucks with circulating water jackets.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, PTFE, PFA). For example, the dynamic filtration module includes at least one support rod or roller with a mechanically flat contact surface to stabilize the movement of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to an active target area of the filter membrane. For example, the at least one output head is a tube or slot die.

In some embodiments, the dynamic filtration module includes at least one additional input line for supplying wash buffer through a coaxial output head, a separate monoaxial output head, a separate slot die output head, or a slot die output head with multiple openings. additionally includes

In some embodiments, the dynamic filtration module may include elements known in the coatings and converting industry, such as, but not intended to be limiting, active or passive edge guides, tension regulators (eg, dancers), brakes and tension detectors; or any combination thereof.

In embodiments, a dynamic filtration module includes a vacuum system in continuity with a membrane support structure to apply a negative pressure across an active target area of a filter membrane, wherein the negative pressure causes active transport of the filter membrane across the membrane support structure. and allow collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. In aspects described herein, during purification by dynamic filtration, the filtrate comprising biological products is fed under negative pressure to a vacuum collection vessel capable of collecting about 50 mL to about 100 L. For example, a vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 50 L.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process includes a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced.

The process described herein involves continuously passing the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising a biological product. For example, separating a solution into two or more fractions may include at least one fraction comprising biological products, and at least one other fraction comprising minor impurities. As described herein, the first module includes an affinity-based TFF purification device. "Affinity-based TFF purification devices" utilize molecular structural binding interactions (e.g., ligand-receptor interactions) in which a selective surface-anchored ligand recognizes and binds to a biological product to be purified with at least one tangential flow filtration system. refers to a purification technology based on For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet.

In embodiments, the affinity-based TFF purification device further comprises a suspension of resin beads. The surface of the resin beads is linked to, for example and without limitation, protein A, protein G, protein L, antigenic protein, protein, receptor, antibody, or aptamer. Continuous purification of biological products (eg, monoclonal antibodies) with affinity resin beads avoids cumbersome processing steps of conventional affinity column chromatography (eg, Protein A affinity chromatography).

In embodiments, the diameter of the resin beads of the affinity-based TFF purification device is between about 10 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of the resin beads may be about 1% by weight to about 20% by weight. In another example, the binding capacity of resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In another example, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

The at least one tangential flow filtration system of the affinity-based TFF purification device may refer to, for example, a tangential flow, high performance tangential flow, or cross-flow filtration system having a flat or hollow fiber membrane filtration configuration. In some examples, the tangential flow filtration system includes a hollow fiber membrane filter. In aspects, the hollow fiber membrane material is selected from PES, modified PES (mPES), or mixed cellulose esters (MCE). In some aspects, hollow fiber membranes can be charged (eg, positively or negatively charged) or uncharged. In another aspect, the pore size of the hollow fiber membrane is selected from the range of about 10 kDa to about 1 μm. In another aspect, the inner diameter of the hollow fiber membrane is selected from the range of about 0.5 mm to about 5 mm.

The processes described herein also include a biological product from at least one first outlet of the first module into a second module having at least one inlet for receiving flow from the at least one first outlet of the first module. wherein the second module comprises a charge-based TFF purification device. As used herein, a “charge-based TFF purification device” refers to purifying a biological molecule based on its surface charge, ionic properties, electrostatic interactions, or isoelectric point, eg, using at least one tangential flow filtration system. includes doing As described herein, charge-based TFF purification devices include positive charge-based TFF purification devices, negative charge-based TFF purification devices, or combinations thereof. For example, the second module has at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet.

In embodiments, the charge-based TFF purification device further comprises a suspension of resin beads. For example, the surface of the resin beads may include cationic or anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based purification or negative charge-based purification, respectively. Continuous purification of biological products (eg, monoclonal antibodies) with ionic resin beads avoids cumbersome processing steps of conventional ion exchange column chromatography (eg, cation exchange or anion exchange chromatography).

In embodiments, the diameter of the resin beads of the charge-based TFF purification device is between about 10 microns and about 200 microns. The diameter of the resin beads may vary depending on the biological product to be purified and the flow rate of the process. In addition, the concentration of the resin beads may be in the range of 0.01% by weight to 25% by weight. For example, the concentration of the resin beads may be about 1% by weight to about 20% by weight. In another example, the charge or electrostatic association capacity of resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof.

The at least one tangential flow filtration system of the charge-based TFF purification device may refer to, for example, a tangential flow, high performance tangential flow, or cross-flow filtration system having a flat or hollow fiber membrane filtration geometry. In some examples, the tangential flow filtration system includes a hollow fiber membrane filter. In aspects, the hollow fiber membrane material is selected from PES, modified PES (mPES), or mixed cellulose esters (MCE). In some aspects, hollow fiber membranes can be charged (eg, positively or negatively charged) or uncharged. In another aspect, the pore size of the hollow fiber membrane is selected from the range of about 10 kDa to about 1 μm. In another aspect, the inner diameter of the hollow fiber membrane is selected from the range of about 0.5 mm to about 5 mm.

In embodiments described herein, the resin beads of one or both of the first (affinity-based TFF purification) and/or second (charge-based TFF purification) modules are recycled and reused. For example, the beads can be reused at least 2, 3, 4, or more times to purify a biological product.

Alternatively, the processes described herein may direct the biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module. wherein the second module comprises a free flow electrophoresis device. A free flow electrophoresis device having a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and an aqueous ionic solution is a charge for purifying a biological product (e.g., a monoclonal antibody). based magnetic purification module(s) may be used in place of or in addition to it.

For example, the solution contacting surfaces of the two parallel plates include glass, ceramic, plastic, or any combination thereof. In some instances, an aqueous ionic solution may generate a pH gradient. In another example, an aqueous ionic solution may impart a constant pH.

In embodiments, a free flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module may have a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (eg, the approximate pH gradient may range from about 2 to about 10); and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separating channel (e.g., the fine pH gradient is a pH of about 5 to about 8). and at least one second fluid element including a range). For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In another embodiment, a free flow electrophoretic device includes an electric field or electric field gradient perpendicular to the direction of fluid flow and a fluidic channel created between two parallel plates to operate in a bandelectrophoresis or charge separation mode of operation, wherein the pH There is at least one fluid element with no gradient. For example, an isoelectric point-based fluid purification module includes a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7). at least one first fluid element; and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (eg, less than pH 7). .

In another embodiment, a free flow electrophoretic device includes a fluid channel created between two parallel plates to operate in an isokinetic electrophoretic mode of operation, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a spacer solution (e.g. , NaCl solution) and at least one fluidic element comprising both an acidic pH gradient and a basic pH gradient.

In another embodiment, the isoelectric point-based fluid purification module includes at least one first free flow electrophoresis device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and 2 at least one second free flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, wherein each element is connected in series; can operate in an independent operating mode that enables For example, at least one first free-flow electrophoresis device may operate in an isoelectric focusing mode and at least one second free-flow electrophoresis device may operate in an isokinetic mode to increase separation resolution.

In another embodiment, an isoelectric based fluid purification module includes at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; A fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g., in the pH range of about 2 to about 10). at least one second free flow electrophoresis device comprising; and a fluid channel created between the two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. and at least one third free flow electrophoresis device. For example, adding a pH gradient across the main separation channel using additional subsequent fluidic devices or chips comprising a fluidic channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow. (e.g., pH range of about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (eg, a platinum wire electrode) to function as an anode or cathode.

In embodiments, back pressure within an isoelectric point-based fluid purification device depends on channel shape and dimensions, inlet and outlet openings and/or tubing diameters, and input flow rate. For example, the back pressure ranges from about 0.5 psi to about 10 psi. In some examples, the back pressure is controlled by, for example, without intending to limit, a needle valve.

In embodiments, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar.

In embodiments, the isoelectric point-based fluid purification device further includes an active cooling system or heat sink (e.g., a Peltier element, a thermal chuck with a circulating water/propylene glycol jacket) to enable temperature control and Joule heat dissipation. do. For example, the active cooling system can control cooling and/or heat dissipation in the range of about 4°C to about 50°C, preferably 4°C to about 37°C. Ideally, the temperature is maintained between about 10° C. and about 25° C. when isolating biological products (eg, monoclonal antibodies). For example, an active cooling system includes an aluminum thermal chuck with a cooled circulating water/propylene glycol jacket.

In embodiments, an isoelectric point based fluid purification module includes at least one buffer or amphoteric electrolyte system.

In embodiments, an isoelectric point based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper functioning in contact with an anode or cathode, for example phosphoric acid and sodium hydroxide, respectively. In another embodiment, at least one electrode solution is configured to contact the anode or cathode to enable proper function, such as Tris buffered saline, flowing through the main separator channel, the anode channel, and the cathode channel. Contains an amphoteric electrolyte solution.

In embodiments, an isoelectric based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is arranged in-line. In some examples, the at least one sensor or detector includes, but is not limited to, a flow sensor, temperature sensor, conductivity sensor, pH sensor, refractive index detector, UV detector, or back pressure sensor.

In embodiments, an isoelectric point-based fluid purification module includes at least one liquid circuit breaker or disconnects downstream of the device and upstream of at least one in-line sensor or detector to perform sensing or detection in a voltage-free solution. make it possible

The presently claimed process is compatible with current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. It offers many advantages over For example, without intending to be limiting, the processes described herein can reduce production facility footprint, facility build-up, when compared to existing approaches to making batch, single use, or semi-continuous monoclonal antibodies while maintaining throughput and yield. and a continuous bioprocess for purifying monoclonal antibodies that significantly reduces the time required for validation, costs associated with facility construction, and capital equipment expenditure. Continuous bioprocessing as described herein, due to its ability to operate continuously, requires large process equipment - the size of which is large bioreactors - for the centrifugation, depth filtration, and column chromatography steps of existing downstream bioprocessing. Volume dependent - allows for the use of smaller and streamlined equipment (eg, smaller bioreactor volume and downstream bioprocess equipment) since no volume dependent is required. Additionally, smaller and streamlined equipment that operates continuously allows the use of much smaller bioreactor(s) that produce monoclonal antibodies at steady state. A continuous bioprocess as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and biological product loss when compared to existing monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems having a footprint that occupies significantly fewer square feet than current technologies without sacrificing product throughput or yield on a kilograms per year basis.

Advantages of the processes and methods described herein include the ability to remove large impurities (eg, cells, cell debris, and aggregates) without membrane fouling or clogging. For example, clarification of cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or blockage of the filter membrane, making such methods unsuitable for long-term continuous processes. making it unsuitable as a means for the continuous removal of large impurities from heterogeneous mixtures containing biological products through In contrast, the dynamic filtration devices described herein enable continuous removal of large impurities from heterogeneous mixtures containing biological products without fouling the membranes, as the active target area of the filter membrane is continuously renewed. In addition, since the entire process of producing and purifying the biological product can be continuous, and a flow rate in the range of about 0.1 mL / min to about 50 mL / min can be maintained throughout the process, the process equipment and overall process footprint ) can occupy a much smaller footprint than current standard processes without sacrificing product throughput or yield on a kilogram/year basis. For example, the processes for producing and purifying monoclonal antibodies described herein operate in footprints occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processing require at least 200,000 square feet. For example, the flow rate of a process to purify a biological product ranges from about 1 mL/min to about 10 mL/min. In some instances, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL/min to about 50 mL/min. In another example, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor discharge line. In another example, a process is provided wherein the flow rate of the step of continuously delivering the filtrate to the first module ranges from about 0.1 mL/min to about 50 mL/min. In another example, a process is provided wherein the flow rate of the step of continuously transferring a fraction comprising biological products from the first outlet to the second module ranges from about 0.1 mL/min to about 50 mL/min.

An important advantage of the processes and methods using resin beads (eg, agarose) described herein is that these systems can be used with existing stationary phase or packed resin columns (eg, standard for chromatography) is not required. For example, such a system provides for recycling and/or regeneration of resin beads to create an infinite surface area of resin beads during operation, resulting in a continuous and cost effective method. In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, the resin beads used during the purification of biological products as described herein are continuously recycled and regenerated so that the flow from the previous step in either the dynamic filtration module or the purification module is not stopped without interrupting the flow from the bioreactor discharge line. can accommodate In other words, because the modules described in the present invention pass through these steps sequentially, they do not need to be left idle for sterilization, regeneration, and/or recycling after execution. The method requires several packed layers to accommodate a continuous input flow and enable regeneration and/or recycling of columns that have reached full capacity, as current column chromatography methods have limited column capacity limitations due to resin packing constraints. It differs from current continuous chromatography methods in that column switching of columns is required. Another advantage of the methods described herein includes that the resin beads do not pack in the stationary phase, but rather the resin beads move. The mobility of these beads increases the surface area of the resin beads available for bonding or association since substantially more of the resin bead surface is exposed and free to bond. Additionally, the resin beads within the packed column are exposed to high pressure differentials to create flow through the column, which damage is one of the reasons the column shortens its desired life. The mobile resin beads of the presently described invention are subjected to substantially lower pressures and thus are much softer to the fragile beads, thereby extending their lifespan. Additionally, this mobility increases the possibility that the resin beads will complete regeneration and return to their initial state. This also makes the method described herein more cost effective because the resin is used more efficiently.

An important advantage of the processes and methods using free flow electrophoresis described herein is that the system represents a "product loss free" process, i.e., the separation is dependent on the physicochemical properties of the target biological product in an aqueous solution and is dependent on the electrical field. The point is that the product does not have to interact with the resin or other purifying moiety because it occurs via interaction. Another advantage is observed in the resolution of this approach, as compared to conventional ion exchange chromatography, theoretically a higher purity product can be obtained. In addition, separation based on essential physiochemical properties can be performed on proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentivirus, but extends the usefulness of this approach for the purification of a variety of biological products, including but not limited to.

In addition, the modular approach provides flexibility in process design to accommodate a wide range of biological products.

dynamic filtration module

Provided herein is a dynamic filtration module for continuously removing large impurities from biological products in a heterogeneous mixture, e.g., cells, cell debris, and aggregates from a heterogeneous mixture derived from a bioreactor operating at steady state. By removing the filtrate containing biological products is produced (Figs. 6a to 6b and 7a to 7b). Unlike the static filtration methods commonly used in the art, the components of a dynamic filtration module move in a coordinated manner so that filtration can continuously occur in new, unused target areas of the filter membrane (e.g., the entire process Depending on the flow rate of the membrane moves or advances). This prevents membrane fouling or clogging and allows control of filter cake packing and thickness during operation.

The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, at least one vacuum line, a vacuum system, and at least one vacuum collection vessel. 6a to 6b and 7a to 7b, the supply reel includes a filter membrane disposed on a filter membrane roll, the filter membrane being supported by two mechanically flat support rods, the opening Passes over a mechanically flat membrane support structure comprising a. As the heterogeneous mixture is delivered from the output head to the active target area of the filter membrane, the filter membrane continues to move, advancing the filter membrane towards the collection reel, while the openings in the membrane support structure and the continuous vacuum line maintain negative pressure, thereby reducing the cell , cell debris, and aggregates are allowed to be separated and removed to produce a filtrate containing biological products.

The dynamic filtration module removes large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture without using centrifugation, depth filtration, static filtration, or any combination thereof to remove biological products and associated small particles. impurities (e.g., host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, undesirable nucleic acids or oligonucleotides, salts, buffer components, surfactants, sugars , metal contaminants, leachates, media components, and/or naturally occurring organic molecules associated therewith).

The dynamic filtration modules described herein provide a small footprint and include tubing, connectors, membrane support structures, and filter membranes (e.g., appropriate material selection for polymer type, pore size).

The dynamic filtration module includes a rolled filter membrane extending between a supply reel and a collection reel, the filter membrane having an active target area configured to receive the heterogeneous mixture. For example, the filter membrane of the filter membrane roll is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), or a suitable material including, but not limited to, hydrophilic PTFE.

In embodiments, the pore size of the rolled filter membrane varies depending on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the rolled filter membrane has a pore size in the range of 0.2 μm to about 0.45 μm.

In embodiments, the width of the filter membrane roll is between about 10 mm and about 600 mm. For example, the width of a filter membrane roll may vary depending on factors such as the size of the dynamic filtration system and the size of the membrane support structure.

In embodiments, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts with a prefabricated roll and continues to an initially empty collection roll to create a reel-to-reel system. In operation, the inhomogeneous mixture is transferred from the output head to a new unused area of the filter membrane created by the transport of the filter membrane as the filter membrane moves at an appropriate speed from the supply reel to the collection reel to collect the used portion of the filter membrane. (herein also referred to as the active target area) is applied sequentially. For example, feed reel movement is controlled by a servo motor coupled with a gearbox that limits revolutions per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collecting reel movement is controlled by a servo motor coupled with a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. Additionally, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure consistent speed with the constantly changing diameter of the filter membrane rolls on both the feed and collect reels during operation. For example, the supply reel and the collection reel run in the same direction at the same speed. Other methods of transporting the filter membrane from the supply reel to the collection reel can be considered by those skilled in the coating and converting industries.

In embodiments, the filter membrane transport rate within the dynamic filtration module is selected to enable a high flow rate (high throughput) while maintaining a high yield (recovery). For example, the transport speed of the filter membrane ranges from about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1 mm/sec to about 10 mm/sec.

The dynamic filtration module also includes a membrane support structure ( FIG. 8 ) for supporting the active target area of the filter membrane when the filter membrane is subjected to negative pressure. The membrane support structure is located between the supply reel and the collection reel, has a mechanically flat contact surface derived from a material with a low static friction coefficient (eg PTFE), and has an opening continuous with the vacuum line. For example, a “mechanically smooth contact surface” as used herein refers to a surface that has a low static coefficient of friction, particularly when wet, that creates a low frictional force opposing the transport of the filter membrane. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, the lower the value the smoother the surface. Also, because rough surfaces have greater friction between surfaces than smooth surfaces, mechanically smooth contact surfaces as used herein refer to surfaces with low friction (i.e., low static coefficient of friction).

In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (eg, a mesh, at least one slot, at least one hole, or any combination thereof). The opening may also include regularly spaced elements, for example, the opening may include a series of equally spaced parallel slots. Additionally, the opening may comprise a grate (eg, a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, where each grate is vertical. The opening may be a collection of irregular or regular elements (eg a series of parallel slots). The openings may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (eg, mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without intending to be limiting, the openings may include a mesh between about 0.25 mm and about 5 mm thick.

In addition, a connection with at least one vacuum collection vessel for corresponding temperature control of the membrane support structure and evaporative cooling is also provided, such that a biological product (e.g., a protein or fragment thereof (polypeptide), an antibody or fragment thereof) is provided. , cytokines, chemokines, enzymes, or growth factors) to prevent clogging, contamination, solution freezing, change in solution viscosity, and denaturation (or precipitation). The temperature control mechanism maintains a temperature between about 4°C and about 37°C. For example, during antibody purification, the temperature control mechanism maintains a temperature between about 15°C and about 37°C. Exemplary temperature control mechanisms include, but are not limited to, single loop controllers, multiple loop controllers, closed loop controllers, PID controllers, Peltier elements, resistive heating elements, and/or thermal chucks with circulating water jackets.

In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static coefficient of friction (eg, PTFE, PFA). For example, as used herein, a “mechanically smooth contact surface” refers to a surface that has a low static coefficient of friction, resulting in a low frictional force opposing the transport of the filter membrane. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, the lower the value the smoother the surface. Also, because rough surfaces have greater friction between surfaces than smooth surfaces, mechanically smooth contact surfaces as used herein refer to surfaces with low friction (i.e., low static coefficient of friction). Alternatively, the at least one support rod may further comprise a bearing, for example a sleeve bearing with a mechanically flat contact surface to reduce friction and tension on the filter membrane. Additionally, at least one support rod or roller having a mechanically flat contact surface can be rotated to reduce tension on the filter membrane.

In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to an active target area of the filter membrane. For example, the at least one output head is a tube or slot die.

Within the dynamic filtration module, the input flow rate matches the exit rate of a bioreactor operating at steady state, where the exit rate corresponds to a significantly higher throughput (e.g., conventional biopharmaceutical manufacturing throughput on a kilogram/year basis). throughput that matches or exceeds). In a specific example, several heads may be used to manage the flow rate, as well as xy rastering or rθ rastering heads with or without movement along the z-axis.

The dynamic filtration module includes the negative pressure of the vacuum system, and as described herein, pressure values can be selected to enable efficient filtration while maintaining desired filter membrane transport mobility to achieve high throughput and yield. In embodiments, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

In some embodiments, a wash zone is provided in addition to and subsequent to the feed zone (eg, bioreactor effluent solution input line and output head distribution zone or filter membrane active target zone). The wash zone includes a wash buffer supplied from an additional input line through a coaxial output head, a separate monoaxial output head, a separate slot die output head, or a slot die output head with multiple openings.

In some embodiments, the dynamic filtration module may include elements known in the coatings and converting industry, such as, but not intended to be limiting, active or passive edge guides, tension regulators (eg, dancers), brakes and tension detectors; or any combination thereof.

In embodiments, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration uses at least two separate rolled filter membranes of different pore sizes. It includes multi-stage filtration. For example, such a multi-stage dynamic filtration process includes a large pore size (eg, 0.2 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (eg, 0.2 μm). ) at least one first dynamic filtration device having a rolled filter membrane, whereby a filtrate comprising biological products is produced.

As described herein, dynamic filtration modules provide comparable or higher yields of biological product on a kilograms per year basis than standard purification (centrifugation) processes. The dynamic filtration module also allows feeding to the next step in at least one vacuum collection vessel that is under negative pressure or can be in equilibrium with atmospheric pressure. selected materials included in the dynamic filtration module include connectors, tubing, filter membranes, membrane support structures, and vacuum collection vessel(s), all of which alone or in combination minimize friction and yield loss due to protein adsorption; known to those skilled in the art.

Unlike the static filtration methods commonly used in the art, the components of a dynamic filtration module move in a coordinated manner to enable continuous, unobstructed, uncontaminated filtration (e.g., from the input line The membrane moves or advances depending on the flow rate of the heterogeneous mixture).

Affinity-based magnetic purification module with loop conveyor system

In the present specification, an affinity-based magnetic purification module for separating a heterogeneous mixture into two or more fractions is provided, wherein at least one fraction contains a biological product. The module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady state operation. can be consistent and consistent over time. In addition, the affinity-based magnetic purification module includes a suspension of affinity magnetic resin beads, wherein the surface of the magnetic resin beads is, without limitation, configured to selectively bind to the biological product: protein A, protein G, Linked to protein L, antigenic protein, protein, receptor, antibody, or aptamer.

The affinity-based magnetic purification module continuously receives a heterogeneous mixture comprising a biological product, and then the resulting heterogeneous mixture comprising a biological product, affinity magnetic resin beads, a buffer, or any combination thereof. a loop conveyor system comprising at least two transport containers filled with affinity magnetic resin beads configured to transport; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable cleaning; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable elution of the biological product; at least one external magnetic field enabling recycling of the magnetic resin beads; at least one bind/wash buffer system; at least one low pH elution buffer system; at least one magnetic resin bead regeneration buffer system; at least one aspirator system to remove waste liquid from the at least two transport vessels; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the affinity-based magnetic purification module (FIGS. 14-15) enables automated and continuous biological magnetic purification compared to batch or semi-continuous processes, and provides a small footprint.

The magnetic field strength and field effect depend on the transport vessel wall thickness and material type, proximity to the transport vessel wall on the loop conveyor track, magnetic resin bead size, concentration, saturation magnetization and magnetic susceptibility, and Depends on solution viscosity. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). For example, the magnetic field is created by permanent magnets (eg neodymium magnets). The permanent magnet may be located within 5 mm, preferably within 1 mm, of the vessel wall. In another example, the magnetic field is generated by an electromagnet. In another example, mixing of the magnetic resin beads may be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

As described herein, the affinity-based magnetic purification module includes a suspension of magnetic resin beads, the concentration of which is dependent on the desired binding capacity or desired solution viscosity. Alternatively, the affinity-based magnetic purification module magnetic resin bead size can be scaled from microns to submicrons, e.g., enabling appropriate fluid dynamics for affinity interactions and equilibrium, such as solution viscosity dependence and surface ligand density dependence, respectively. binding capacity requirements, which are a function of the surface area-to-volume ratio required to

The number and size of transport vessels depends on input flow rate, magnetic resin bead binding capacity, and binding equilibration time. The material and wall thickness of the transport container depends on the strength and proximity of the magnetic field.

The material selection for the magnetic resin beads of the affinity-based magnetic purification module is important to provide negligible leachables and robust stability enabling recycling and reuse. Magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

Binding/washing buffers depend on the desired biological product (eg monoclonal antibody) and minor impurities to be removed. Other considerations for binding/washing buffers are also taken into account in binding/washing buffers, including pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts. Additionally, additional transport containers and repeated conveyor track positions may be required for effective cleaning.

The elution buffer depends on the binding affinity (eg, strength of non-covalent interaction) between the magnetic resin bead surface ligands and the desired biological product (eg, monoclonal antibody). For example, the elution buffer can be adjusted according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different elution buffer compositions (e.g., to increase yield, this may include additional transport containers and repeated conveyors). track position (which may be required) may be varied. Additionally, additional transport containers and repeated conveyor track locations may be required for effective elution.

The dwell time of each stage in the conveyor track progression depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, viral inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The affinity-based magnetic purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the affinity-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ. more efficiently and enable continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Affinity-based magnetic purification module with pick-and-place robot system

In the present specification, an affinity-based magnetic purification module for separating a mixture into two or more fractions is provided, wherein at least one fraction contains a biological product. The module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady state operation. can be consistent and consistent over time. In addition, the affinity-based magnetic purification module includes a suspension of affinity magnetic resin beads, wherein the surface of the magnetic resin beads is, without limitation, configured to selectively bind to the biological product: protein A, protein G, Linked to protein L, antigenic protein, protein, receptor, antibody, or aptamer.

The affinity-based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, affinity magnetic resin beads, a buffer, or any combination thereof. a pick-and-place robot system comprising at least two transport containers filled with the constructed affinity magnetic resin beads; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable cleaning; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable elution of the biological product; at least one external magnetic field enabling recycling of the magnetic resin beads; at least one bind/wash buffer system; at least one low pH elution buffer system; at least one magnetic resin bead regeneration buffer system; at least one aspirator system to remove waste liquid from the at least two transport vessels; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the affinity-based magnetic purification module (FIG. 18) enables automated and continuous biological magnetic purification compared to batch or semi-continuous processes and provides a small footprint.

The magnetic field strength and field effect depend on the transport container wall thickness and type of material, the proximity to the placed transport container wall, the magnetic resin bead size, concentration, saturation magnetization and susceptibility, and solution viscosity. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). For example, the magnetic field is created by permanent magnets (eg neodymium magnets). The permanent magnet may be located within 5 mm, preferably within 1 mm, of the vessel wall. In another example, the magnetic field is generated by an electromagnet. In another example, mixing of the magnetic resin beads may be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

As described herein, the affinity-based magnetic purification module includes a suspension of magnetic resin beads, the concentration of which is dependent on the desired binding capacity or desired solution viscosity. Alternatively, affinity-based magnetic purification module magnetic resin bead sizes can be scaled from microns to submicrons, e.g., enabling appropriate fluid dynamics for equilibrium and affinity interactions, such as solution viscosity dependence and surface ligand density dependence, respectively. binding capacity requirements, which are a function of the surface area-to-volume ratio required to

The number and size of transport vessels depends on input flow rate, magnetic resin bead binding capacity, and binding equilibration time. The material and wall thickness of the transport container depends on the strength and proximity of the magnetic field.

The material selection for the magnetic resin beads of the affinity-based magnetic purification module is important to provide negligible leachables and robust stability enabling recycling and reuse. Magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Binding/washing buffers depend on the desired biological product (eg monoclonal antibody) and minor impurities to be removed. Other considerations for binding/washing buffers are also taken into account in binding/washing buffers, including pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts. Additionally, additional transport containers and repeated pick-and-place locations may be required for effective cleaning.

The elution buffer depends on the binding affinity (eg, strength of non-covalent interaction) between the magnetic resin bead surface ligands and the desired biological product (eg, monoclonal antibody). For example, the elution buffer can be adjusted according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different elution buffer compositions (e.g., to increase yield, this may include additional transport containers and repeated conveyors). track position (which may be required) may be varied. Additionally, additional transport containers and repeated pick-and-place locations may be required for effective elution.

The residence time of each step in the transport vessel progression through the pick and place process depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, viral inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The affinity-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the affinity-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ to more efficiently process the resin. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Positive Charge Based Magnetic Purification Module with Loop Conveyor System

As described herein, a positive charge based magnetic purification module is included for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation. In addition, the positive charge-based magnetic purification module includes a suspension of cationic magnetic resin beads, wherein the surface of the magnetic resin beads includes cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

The positive charge based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, cationic magnetic resin beads, a buffer, or any combination thereof. a loop conveyor system comprising at least two transport containers filled with cationic magnetic resin beads; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable cleaning; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable dissociation of the biological product; at least one external magnetic field enabling recycling of the magnetic resin beads; at least one dissociation/wash buffer system, at least one dissociation buffer system; at least one magnetic resin bead regeneration buffer system; at least one aspirator system to remove waste liquid from the at least two transport vessels; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the positive charge based magnetic purification module ( FIGS. 16-17 ) enables automated and continuous biological magnetic purification compared to batch or semi-continuous processes and provides a small footprint.

The magnetic field strength and field effect depend on the transport vessel wall thickness and material type, proximity to the transport vessel wall on the loop conveyor track, magnetic resin bead size, concentration, saturation magnetization and susceptibility, and solution viscosity. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). For example, the magnetic field is created by permanent magnets (eg neodymium magnets). The permanent magnet may be located within 5 mm, preferably within 1 mm, of the vessel wall. In another example, the magnetic field is generated by an electromagnet. In another example, mixing of the magnetic resin beads may be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

As described herein, a positive charge-based magnetic purification module includes a suspension of magnetic resin beads, the concentration of which depends on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, positive charge-based magnetic purification module magnetic resin bead sizes range from microns to submicrons, e.g., equilibrium such as solution viscosity dependence and surface charge density dependence, respectively, and appropriate fluid dynamics for charge or electrostatic interactions. Depends on the charge or electrostatic association capacity requirements, which are a function of the surface area to volume ratio required to enable

The number and size of transport vessels depends on the input flow rate, the magnetic resin bead charge or electrostatic association capacity, and the association equilibration time. The material and wall thickness of the transport container depends on the strength and proximity of the magnetic field.

The material selection for the magnetic resin beads of the positive charge-based magnetic purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within positive charge-based magnetic purification modules, cationic surface selection is an important consideration, and may include cationic polymers, net positively charged peptides or proteins, and amine functional groups. In addition, the selection of cationic surfaces is based on achieving appropriate electrostatic interactions and stability between the positively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/washing buffer of the positive charge based magnetic purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional transport containers and repeated conveyor track positions may be required for effective cleaning.

The dissociation buffer of the positive charge-based magnetic purification module depends on the strength of the electrostatic interaction between the cationic magnetic resin bead surface functional group and the desired biological product (eg, monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, which may include additional transport vessels and repeated conveyors). track position (which may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional transport containers and repeated conveyor track positions may be required for effective dissociation.

The residence time of each step in the conveyor track progression of the positive charge-based magnetic purification module depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, viral inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The positive charge based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the positive charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Positive charge-based magnetic purification module with pick-and-place robotic system

As described herein, a positive charge based magnetic purification module is included for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation. In addition, the positive charge-based magnetic purification module includes a suspension of cationic magnetic resin beads, wherein the surface of the magnetic resin beads includes cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

The positive charge based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, cationic magnetic resin beads, a buffer, or any combination thereof. a pick-and-place robotic system comprising at least two transport containers filled with cationic magnetic resin beads; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable cleaning; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable dissociation of the biological product; at least one external magnetic field enabling recycling of the magnetic resin beads; at least one dissociation/wash buffer system, at least one dissociation buffer system; at least one magnetic resin bead regeneration buffer system; at least one aspirator system to remove waste liquid from the at least two transport vessels; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the positive charge-based magnetic purification module (FIG. 19) enables automated and continuous biological magnetic purification compared to batch or semi-continuous processes and provides a small footprint.

The magnetic field strength and field effect depend on the transport vessel wall thickness and material type, proximity to the transport vessel wall on the loop conveyor track, magnetic resin bead size, concentration, saturation magnetization and susceptibility, and solution viscosity. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). For example, the magnetic field is created by permanent magnets (eg neodymium magnets). The permanent magnet may be located within 5 mm, preferably within 1 mm, of the container wall. In another example, the magnetic field is generated by an electromagnet. In another example, mixing of the magnetic resin beads may be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

As described herein, a positive charge-based magnetic purification module includes a suspension of magnetic resin beads, the concentration of which depends on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, positive charge-based magnetic purification module magnetic resin bead sizes range from microns to submicrons, e.g., equilibrium such as solution viscosity dependence and surface charge density dependence, respectively, and appropriate fluid dynamics for charge or electrostatic interactions. Depends on the charge or electrostatic association capacity requirements, which are a function of the surface area to volume ratio required to enable

The number and size of transport vessels depends on the input flow rate, the magnetic resin bead charge or electrostatic association capacity, and the association equilibration time. The material and wall thickness of the transport container depends on the strength and proximity of the magnetic field.

The material selection for the magnetic resin beads of the positive charge-based magnetic purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within positive charge-based magnetic purification modules, cationic surface selection is an important consideration, and may include cationic polymers, net positively charged peptides or proteins, and amine functional groups. In addition, the selection of cationic surfaces is based on achieving appropriate electrostatic interactions and stability between the positively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/washing buffer of the positive charge based magnetic purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional transport containers and repeated pick-and-place locations may be required for effective cleaning.

The dissociation buffer of the positive charge-based magnetic purification module depends on the strength of the electrostatic interaction between the cationic magnetic resin bead surface functional group and the desired biological product (eg, monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, which may include additional transport vessels and repeated conveyors). track position (which may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional transport containers and repeated pick-and-place locations may be required for effective dissociation.

In the transport vessel progress through the pick-and-place process of the positive charge-based magnetic purification module, the residence time of each stage varies depending on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, viral inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The positive charge-based magnetic purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the positive charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Negative Charge Based Magnetic Purification Module with Loop Conveyor System

As described herein, a negative charge based magnetic purification module is included for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation. In addition, the negative charge-based magnetic purification module includes a suspension of anionic magnetic resin beads, wherein the surface of the magnetic resin beads includes anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

The negative charge based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, anionic magnetic resin beads, a buffer, or any combination thereof. a loop conveyor system comprising at least two transport containers filled with anionic magnetic resin beads; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable cleaning; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable dissociation of the biological product; at least one external magnetic field enabling recycling of the magnetic resin beads; at least one dissociation/wash buffer system; at least one dissociation buffer system; at least one magnetic resin bead regeneration buffer system; at least one aspirator system to remove waste liquid from the at least two transport vessels; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the negative charge based magnetic purification module ( FIGS. 16-17 ) enables automated and continuous biological magnetic purification compared to batch or semi-continuous processes, and provides a small footprint.

The magnetic field strength and field effect depend on the transport vessel wall thickness and material type, proximity to the transport vessel wall on the loop conveyor track, magnetic resin bead size, concentration, saturation magnetization and susceptibility, and solution viscosity. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). For example, the magnetic field is created by permanent magnets (eg neodymium magnets). The permanent magnet may be located within 5 mm, preferably within 1 mm, of the container wall. In another example, the magnetic field is generated by an electromagnet. In another example, mixing of the magnetic resin beads may be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

As described herein, a negative charge based magnetic purification module includes a suspension of magnetic resin beads, the concentration of which depends on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, negative charge-based magnetic purification module magnetic resin bead sizes range from microns to submicrons, e.g., equilibrium such as solution viscosity dependence and surface charge density dependence, respectively, and appropriate fluid dynamics for charge or electrostatic interactions. Depends on the charge or electrostatic association capacity requirements, which are a function of the surface area to volume ratio required to enable

The number and size of transport vessels depends on the input flow rate, the magnetic resin bead charge or electrostatic association capacity, and the association equilibration time. The material and wall thickness of the transport container depends on the strength and proximity of the magnetic field.

The material selection for the magnetic resin beads of the negative charge-based magnetic purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within negative charge-based magnetic purification modules, anionic surface selection is an important consideration and may include anionic polymers, net negatively charged peptides or proteins, oligonucleotides, and carboxyl functional groups. Also, the selection is based on achieving adequate electrostatic interaction and stability between the negatively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/washing buffer of the negative charge-based magnetic purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional transport containers and repeated conveyor track positions may be required for effective cleaning.

The dissociation buffer of the negative charge-based magnetic purification module depends on the strength of the electrostatic interaction between the anionic magnetic resin bead surface functional group and the desired biological product (eg, monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, which may include additional transport vessels and repeated conveyors). track position (which may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional transport containers and repeated conveyor track positions may be required for effective dissociation.

The residence time of each step in the conveyor track progression of the negative charge-based magnetic purification module depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, viral inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The negative charge-based magnetic purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the negative charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Negative charge-based magnetic purification module with pick-and-place robot system

As described herein, a negative charge based magnetic purification module is included for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation. In addition, the negative charge-based magnetic purification module includes a suspension of anionic magnetic resin beads, wherein the surface of the magnetic resin beads includes anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

The negative charge based magnetic purification module is configured to continuously receive a mixture comprising a biological product and then transport the resulting heterogeneous mixture comprising the biological product, anionic magnetic resin beads, a buffer, or any combination thereof. a pick-and-place robot system comprising at least two transport containers filled with anionic magnetic resin beads; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable cleaning; at least one external magnetic field that attracts and separates the magnetic resin beads from the heterogeneous mixture to enable dissociation of the biological product; at least one external magnetic field enabling recycling of the magnetic resin beads; at least one dissociation/wash buffer system; at least one dissociation buffer system; at least one magnetic resin bead regeneration buffer system; at least one aspirator system to remove waste liquid from the at least two transport vessels; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the negative charge based magnetic purification module ( FIG. 19 ) enables automated and continuous biological magnetic purification compared to batch or semi-continuous processes and provides a small footprint.

The magnetic field strength and field effect depend on the transport vessel wall thickness and material type, proximity to the transport vessel wall on the loop conveyor track, magnetic resin bead size, concentration, saturation magnetization and susceptibility, and solution viscosity. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). For example, the magnetic field is created by permanent magnets (eg neodymium magnets). The permanent magnet may be located within 5 mm, preferably within 1 mm, of the container wall. In another example, the magnetic field is generated by an electromagnet. In another example, mixing of the magnetic resin beads may be achieved by placing the at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

As described herein, a negative charge based magnetic purification module includes a suspension of magnetic resin beads, the concentration of which depends on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, negative charge-based magnetic purification module magnetic resin bead sizes range from microns to submicrons, e.g., equilibrium such as solution viscosity dependence and surface charge density dependence, respectively, and appropriate fluid dynamics for charge or electrostatic interactions. Depends on the charge or electrostatic association capacity requirements, which are a function of the surface area to volume ratio required to enable

The number and size of transport vessels depends on the input flow rate, the magnetic resin bead charge or electrostatic association capacity, and the association equilibration time. The material and wall thickness of the transport container depends on the strength and proximity of the magnetic field.

The material selection for the magnetic resin beads of the negative charge-based magnetic purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within negative charge-based magnetic purification modules, anionic surface selection is an important consideration and may include anionic polymers, net negatively charged peptides or proteins, oligonucleotides, and carboxyl functional groups. Also, the selection is based on achieving adequate electrostatic interaction and stability between the negatively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/washing buffer of the negative charge-based magnetic purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional transport containers and repeated pick-and-place locations may be required for effective cleaning.

The dissociation buffer of the negative charge-based magnetic purification module depends on the strength of the electrostatic interaction between the anionic magnetic resin bead surface functional group and the desired biological product (eg, monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, which may include additional transport vessels and repeated conveyors). track position (which may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional transport containers and repeated pick-and-place locations may be required for effective dissociation.

In the transport vessel progress through the pick-and-place process of the negative charge-based magnetic purification module, the residence time of each stage varies depending on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The negative charge-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the negative charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Affinity-based purification module with mechanical rotation system

In this specification, an affinity-based purification module is provided for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady state operation. can be consistent and consistent over time. The affinity-based purification module also includes a suspension of affinity resin beads, wherein the surface of the resin beads is, without limitation, configured to selectively bind to the biological product: protein A, protein G, protein L, antigen. It is linked to a protein, protein, receptor, antibody, or aptamer.

The affinity-based purification module is a lid system movable along the z-axis having at least one gasket lead, wherein the at least one gasket lid has at least one inlet for introducing gas to control a static head pressure, to equilibrate with atmospheric pressure. at least one vent port to achieve this, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving a filtrate containing a biological product that allows binding, washing of the resin beads, these comprising at least two inlets for introducing a buffer system for dispersing the resin beads to enable elution from, or regeneration thereof; A mechanical rotational system movable in the xy plane, eg, continuously receives a mixture comprising a biological product, and then displaces the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof. a carousel comprising at least two containers filled with resin beads configured to transport; a collection system movable along the z-axis that interfaces with at least one of the at least two vessels of the mechanical rotation system to collect waste, fractions comprising biological products, or any combination thereof; at least one gas; at least one bind/wash buffer system; at least one elution buffer system; at least one resin bead regeneration buffer system; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the affinity-based purification module ( FIGS. 21 and 23 ) enables automated and continuous biological purification compared to batch or semi-continuous processes, and provides a small footprint.

As described herein, affinity-based purification modules include a suspension of resin beads, the concentration of which depends on the desired binding capacity or desired solution viscosity. Alternatively, the affinity-based purification module resin bead size ranges from microns to submicrons, e.g., to enable proper hydrodynamics for equilibrium and affinity interactions, such as solution viscosity dependence and surface ligand density dependence, respectively. It depends on the bonding capacity requirement, which is a function of the required surface area to volume ratio.

The number and size of vessels depends on input flow rate, resin bead binding capacity, and binding equilibration time. The material of the container is selected to limit protein binding.

The material of the filter or filter membrane and the pore size of the vessel depend on the resin bead diameter and the desired biological product. The material of the filter or filter membrane is selected to limit protein binding.

Material selection for the resin beads of the affinity-based purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Binding/washing buffers depend on the desired biological product (eg monoclonal antibody) and minor impurities to be removed. Other considerations for binding/washing buffers are also taken into account in binding/washing buffers, including pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts. Additionally, additional containers and repeated carousel positions may be required for effective cleaning.

The elution buffer depends on the binding affinity (eg, strength of non-covalent interactions) between the resin bead surface ligands and the desired biological product (eg, monoclonal antibody). For example, the elution buffer may be adjusted according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different elution buffer compositions (e.g., to increase yield, this may include additional vessels and repeated carousels). location may be required) may be varied. Additionally, additional containers and repeated carousel positions or additional elution may be required for effective elution.

The residence time of each step in the vessel progression through the rotational process depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The affinity-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the affinity-based purification module uses a mobile affinity resin that can be regenerated and recycled in situ, enabling more efficient use of the resin. , enabling continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Positive Charge Based Purification Module with Mechanical Rotation System

As described herein, a positive charge based purification module is included for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge based purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady. State can be consistent and constant during operation. The positive charge based purification module also includes a suspension of cationic resin beads, wherein the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

The positive charge based purification module is a lead system movable along the z-axis having at least one gasket lead, wherein the at least one gasket lead is in equilibrium with atmospheric pressure, at least one inlet for introducing gas to be able to control the hydrostatic head pressure. at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving a filtrate containing a biological product capable of association, washing the resin beads, from them a lid system movable along the z-axis having at least two inlets for introducing a buffer system that disperses the resin beads to enable their dissociation, or regeneration thereof; A mechanical rotational system movable in the xy plane, eg, continuously receives a mixture comprising a biological product, and then displaces the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof. a carousel comprising at least two containers filled with resin beads configured to transport; a collection system movable along the z-axis that interfaces with at least one of the at least two vessels of the mechanical rotation system to collect waste, fractions comprising biological products, or any combination thereof; at least one gas; at least one association/wash buffer system; at least one dissociation buffer system; at least one resin bead regeneration buffer system; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the positive charge based purification module ( FIGS. 22-23 ) enables automated and continuous biological purification compared to batch or semi-continuous processes and provides a small footprint.

As described herein, positive charge based purification modules include cationic resin beads, the concentration of which is dependent on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, the positive charge based purification module resin bead size ranges from microns to submicrons, e.g., to allow for equilibrium such as solution viscosity dependence and surface charge density dependence and appropriate fluid dynamics for charge or electrostatic interactions, respectively. It depends on the charge or electrostatic association capacity requirements, which are a function of the surface area-to-volume ratio required to

The number and size of vessels depends on input flow rate, resin bead charge or electrostatic association capacity, and association equilibration time. The material of the container is selected to limit protein binding.

The material of the filter or filter membrane and the pore size of the vessel depend on the resin bead diameter and the desired biological product. The material of the filter or filter membrane is selected to limit protein binding.

The material choice for the resin beads of the positive charge-based purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within positive charge-based purification modules, cationic surface selection is an important consideration and may include cationic polymers, net positively charged peptides or proteins, and amine functional groups. In addition, the selection is based on achieving adequate electrostatic interaction and stability between the positively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/washing buffer of the positive charge based purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional containers and repeated carousel positions may be required for effective cleaning.

The dissociation buffer of the positive charge based purification module depends on the strength of the electrostatic interaction between the anionic resin bead surface functional groups and the desired biological product (eg monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, this may include additional vessels and repeated carousels). location may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional containers and repeated carousel positions or additional dissociation may be required for effective dissociation.

The residence time of each step in the vessel progression through the rotational process depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The positive charge-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the positive charge-based purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently, It enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Negative Charge Based Purification Module with Mechanical Rotation System

As described herein, a negative charge based purification module is included for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge based purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady. State can be consistent and constant during operation. The negative charge based purification module also includes a suspension of anionic resin beads, wherein the surface of the resin beads contains anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength.

The negative charge based purification module is a lead system movable along the z-axis having at least one gasket lead, wherein the at least one gasket lead is in equilibrium with atmospheric pressure, at least one inlet for introducing gas to control the static head pressure. at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving a filtrate containing a biological product capable of association, washing the resin beads, from them a lid system movable along the z-axis having at least two inlets for introducing a buffer system that disperses the resin beads to enable their dissociation, or regeneration thereof; A mechanical rotational system movable in the xy plane, eg, continuously receives a mixture comprising a biological product, and then displaces the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof. a carousel comprising at least two containers filled with resin beads configured to transport; a collection system movable along the z-axis that interfaces with at least one of the at least two vessels of the mechanical rotation system to collect waste, fractions comprising biological products, or any combination thereof; at least one gas; at least one association/wash buffer system; at least one dissociation buffer system; at least one resin bead regeneration buffer system; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the negative charge based purification module ( FIGS. 22-23 ) enables automated and continuous biological purification compared to batch or semi-continuous processes and provides a small footprint.

As described herein, negative charge-based purification modules include a suspension of resin beads, the concentration of which depends on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, the negative charge based purification module resin bead size ranges from microns to submicrons, e.g., to allow for equilibrium such as solution viscosity dependence and surface charge density dependence and appropriate fluid dynamics for charge or electrostatic interactions, respectively. It depends on the charge or electrostatic association capacity requirements, which are a function of the surface area-to-volume ratio required to

The number and size of vessels depends on input flow rate, resin bead charge or electrostatic association capacity, and association equilibration time. The material of the container is selected to limit protein binding.

The material of the filter or filter membrane and the pore size of the vessel depend on the resin bead diameter and the desired biological product. The material of the filter or filter membrane is selected to limit protein binding.

The material choice for the resin beads of the negative charge-based purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within the negative charge based purification module, anionic surface selection is an important consideration and may include anionic polymers, net negatively charged peptides or proteins, oligonucleotides, carboxyl functional groups. Also, the selection is based on achieving adequate electrostatic interaction and stability between the negatively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/wash buffer of the negative charge based purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional containers and repeated carousel positions may be required for effective cleaning.

The dissociation buffer of the negative charge-based purification module depends on the strength of the electrostatic interaction between the anionic resin bead surface functional groups and the desired biological product (eg, monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, this may include additional vessels and repeated carousels). location may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional containers and repeated carousel positions or additional dissociation may be required for effective dissociation.

The residence time of each step in the vessel progression through the rotational process depends on the flow rate, equilibration time, and throughput volume. Also, depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The negative charge-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the negative charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Affinity-based purification module with a stepwise linear system

In this specification, an affinity-based purification module is provided for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady state operation. can be consistent and consistent over time. The affinity-based purification module also includes a suspension of affinity resin beads, wherein the surface of the resin beads is, without limitation, configured to selectively bind to the biological product: protein A, protein G, protein L, antigen. It is linked to a protein, protein, receptor, antibody, or aptamer.

The affinity-based purification module includes at least one inlet for introducing gas to control the hydrostatic head pressure, at least one vent port for equilibrating with atmospheric pressure, and at least one inlet for introducing a suspension of resin beads. , at least one inlet for receiving a filtrate containing biological products enabling binding, introducing a buffer system for dispersing the resin beads to enable washing, elution from, or regeneration of the resin beads. at least one gasket lid system having at least two inlets for At least two vessels filled with migratory resin beads configured to continuously receive a mixture comprising a biological product and then to process the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof. A stepwise linear system including; a collection system coupled to at least one of the at least two vessels of the staged linear system to collect waste, fractions comprising biological products, or any combination thereof; at least one gas; at least one bind/wash buffer system; at least one elution buffer system; at least one resin bead regeneration buffer system; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the affinity-based purification module ( FIGS. 24A and 24B ) enables automated and continuous biological purification compared to batch or semi-continuous processes, and provides a small footprint.

As described herein, affinity-based purification modules include a suspension of resin beads, the concentration of which depends on the desired binding capacity or desired solution viscosity. Alternatively, the affinity-based purification module resin bead size ranges from microns to submicrons, e.g., to enable proper hydrodynamics for equilibrium and affinity interactions, such as solution viscosity dependence and surface ligand density dependence, respectively. It depends on the bonding capacity requirement, which is a function of the required surface area to volume ratio.

The number and size of vessels depends on input flow rate, resin bead binding capacity, and binding equilibration time. The material of the container is selected to limit protein binding.

The material of the filter or filter membrane and the pore size of the vessel depend on the resin bead diameter and the desired biological product. The material of the filter or filter membrane is selected to limit protein binding.

Material selection for the resin beads of the affinity-based purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Binding/washing buffers depend on the desired biological product (eg monoclonal antibody) and minor impurities to be removed. Other considerations for binding/washing buffers are also taken into account in binding/washing buffers, including pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts. Additionally, additional containers and repeated carousel positions may be required for effective cleaning.

The elution buffer depends on the binding affinity (eg, strength of non-covalent interactions) between the resin bead surface ligands and the desired biological product (eg, monoclonal antibody). For example, the elution buffer may be adjusted according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different elution buffer compositions (e.g., to increase yield, this may include additional vessels and repeated carousels). location may be required) may be varied. Also, additional elution may be required for effective elution.

Depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The affinity-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the affinity-based purification module uses a mobile affinity resin that can be regenerated and recycled in situ, enabling more efficient use of the resin. , enabling continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Positive Charge Based Purification Module with Stepwise Linear System

Provided herein is a positive charge based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady state operation. can be consistent and consistent over time. The positive charge based purification module also includes a suspension of affinity resin beads, wherein the surface of the resin beads is, without limitation, configured to bind selectively to the biological product: protein A, protein G, protein L, antigenic protein. , linked to proteins, receptors, antibodies, or aptamers.

The positive charge based purification module includes at least one inlet for introducing gas to control the static head pressure, at least one vent port for equilibrating with atmospheric pressure, at least one inlet for introducing a suspension of resin beads, At least one inlet for receiving a filtrate containing biological products, at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. at least one gasket lid system having At least two vessels filled with migratory resin beads configured to continuously receive a mixture comprising a biological product and then to process the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof. A stepwise linear system including; a collection system coupled to at least one of the at least two vessels of the staged linear system to collect waste, fractions comprising biological products, or any combination thereof; at least one gas; at least one bind/wash buffer system; at least one elution buffer system; at least one resin bead regeneration buffer system; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the positive charge based purification module ( FIGS. 24A and 24B ) enables automated and continuous biological purification compared to batch or semi-continuous processes and provides a small footprint.

As described herein, positive charge based purification modules include cationic resin beads, the concentration of which is dependent on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, the positive charge based purification module resin bead size ranges from microns to submicrons, e.g., to allow for equilibrium such as solution viscosity dependence and surface charge density dependence and appropriate fluid dynamics for charge or electrostatic interactions, respectively. It depends on the charge or electrostatic association capacity requirements, which are a function of the surface area-to-volume ratio required to

The number and size of vessels depends on input flow rate, resin bead charge or electrostatic association capacity, and association equilibration time. The material of the container is selected to limit protein binding.

The material of the filter or filter membrane and the pore size of the vessel depend on the resin bead diameter and the desired biological product. The material of the filter or filter membrane is selected to limit protein binding.

The material choice for the resin beads of the positive charge-based purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within positive charge-based purification modules, cationic surface selection is an important consideration and may include cationic polymers, net positively charged peptides or proteins, and amine functional groups. In addition, the selection is based on achieving adequate electrostatic interaction and stability between the positively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/washing buffer of the positive charge based purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional containers and repeated carousel positions may be required for effective cleaning.

The dissociation buffer of the positive charge based purification module depends on the strength of the electrostatic interaction between the anionic resin bead surface functional groups and the desired biological product (eg monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, this may include additional vessels and repeated carousels). location may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional dissociation may be required for effective dissociation.

Depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The positive charge-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the positive charge-based purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently, It enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Negative Charge Based Purification Module with Stepwise Linear System

Provided herein is a negative charge based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, steady state operation. can be consistent and consistent over time. The negative charge based purification module also includes a suspension of affinity resin beads, wherein the surface of the resin beads is, without limitation, configured to bind selectively to the biological product: protein A, protein G, protein L, antigenic protein. , linked to proteins, receptors, antibodies, or aptamers.

The negative charge based purification module includes at least one inlet for introducing gas to control the positive head pressure, at least one vent port for equilibrating with atmospheric pressure, at least one inlet for introducing a suspension of resin beads, At least one inlet for receiving a filtrate containing biological products, at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. at least one gasket lid system having At least two vessels filled with migratory resin beads configured to continuously receive a mixture comprising a biological product and then to process the resulting heterogeneous mixture comprising the biological product, resin beads, buffer, or any combination thereof. A stepwise linear system including; a collection system coupled to at least one of the at least two vessels of the staged linear system to collect waste, fractions comprising biological products, or any combination thereof; at least one gas; at least one bind/wash buffer system; at least one elution buffer system; at least one resin bead regeneration buffer system; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the negative charge based purification module ( FIGS. 25A and 25B ) enables automated and continuous biological purification compared to batch or semi-continuous processes and provides a small footprint.

As described herein, negative charge-based purification modules include a suspension of resin beads, the concentration of which depends on the desired charge or electrostatic association capacity, or the desired solution viscosity. Alternatively, the negative charge based purification module resin bead size ranges from microns to submicrons, e.g., to allow for equilibrium such as solution viscosity dependence and surface charge density dependence and appropriate fluid dynamics for charge or electrostatic interactions, respectively. It depends on the charge or electrostatic association capacity requirements, which are a function of the surface area-to-volume ratio required to

The number and size of vessels depends on input flow rate, resin bead charge or electrostatic association capacity, and association equilibration time. The material of the container is selected to limit protein binding.

The material of the filter or filter membrane and the pore size of the vessel depend on the resin bead diameter and the desired biological product. The material of the filter or filter membrane is selected to limit protein binding.

The material choice for the resin beads of the negative charge-based purification module is important to provide negligible leachables and robust stability to enable recycling and reuse. Resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

Within the negative charge based purification module, anionic surface selection is an important consideration and may include anionic polymers, net negatively charged peptides or proteins, oligonucleotides, carboxyl functional groups. Also, the selection is based on achieving adequate electrostatic interaction and stability between the negatively charged bead surface and the biological product within a defined buffer (pH and ionic strength).

The association/wash buffer of the negative charge based purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Additionally, pH, ionic strength, use of surfactants, and use of organic and/or inorganic salts are also considered in the association/wash buffer. Additionally, additional containers and repeated carousel positions may be required for effective cleaning.

The dissociation buffer of the negative charge-based purification module depends on the strength of the electrostatic interaction between the anionic resin bead surface functional groups and the desired biological product (eg, monoclonal antibody). For example, the dissociation buffer may be modified according to pH, ionic strength, use of surfactants, use of organic and/or inorganic salts, use of different dissociation buffer compositions (e.g., to increase yield, this may include additional vessels and repeated carousels). location may be required) may be varied. In another example, several dissociation buffers with varying pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect. Additionally, additional dissociation may be required for effective dissociation.

Depending on the buffer composition and pH, virus inactivation and removal during the washing step is also taken into account, so that, for example, when purifying monoclonal antibodies, a separate virus inactivation and removal process step will not be necessary.

The negative charge-based purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the negative charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Affinity-based fluid purification module

As provided herein, an affinity-based fluid purification module for separating a mixture into two or more fractions is described, wherein at least one fraction contains a biological product. The affinity-based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, For example, it can be consistent and constant during steady state operation. The affinity-based fluid purification module also includes a suspension of affinity magnetic resin beads, wherein the surface of the magnetic resin beads is, without limitation, configured to selectively bind to the biological product: protein A, protein G, Linked to protein L, antigenic protein, protein, receptor, antibody, or aptamer.

The affinity-based fluid purification module includes at least one equilibration vessel enabling binding of a biological product to a surface of a magnetic resin bead; at least one low pH equilibration vessel allowing debinding of the biological product from the magnetic resin bead surface; At least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to manipulate the flow path of the magnetic resin beads in the heterogeneous mixture, and fire the magnetic resin beads coupled to the biological product. at least one first hybrid cross-flow fluid element or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any of these) comprising a cross-flow channel for separation from small impurities in the homogeneous mixture any combination) (FIG. 28); at least one magnetic field and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to manipulate the flow path of the magnetic resin beads from the biological product, and separating the magnetic resin beads from the biological product. at least one second hybrid cross-flow fluid element or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any combination thereof) comprising a cross-flow channel for ; at least two buffer systems; at least one magnetic resin bead regeneration buffer system; at least one regeneration equilibration vessel configured to enable recycling of the magnetic resin beads; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the affinity-based fluid purification module (FIG. 29) enables continuous biomagnetic purification and provides a small footprint.

The equilibrium vessel volume and agitation capacity of the at least one affinity-based fluid purification module takes into account binding kinetics and input flow rate and throughput, equilibration time and agitation rate enabling magnetic resin bead concentration and binding capacity. Equilibration buffers for affinity-based fluid purification modules depend on the desired biological product (eg, monoclonal antibody) and the small impurities to be removed. Considerations for the buffer include pH, ionic strength, use of surfactants, or use of organic and/or inorganic salts.

The cross-flow channel size of the affinity-based fluid purification module depends on solution viscosity, magnetic resin bead concentration, input flow rate, and throughput volume. Piezoelectric or acoustic actuators allow for desired magnetic resin bead deflection or manipulation of the magnetic resin bead flow path, and a physical position and energy (eg, frequency) that enables a type of piezoelectric crystal. Dielectrophoretic electrodes take into account the optional type and design that enables the desired magnetic resin bead deflection, the number and spacing of electrodes that allow the desired bead deflection, the applied voltage that allows the desired bead deflection, and the electrode material.

The magnetic field strength and field effect depend on the flow rate and magnetic resin bead concentration, size, saturation magnetization and susceptibility, and/or proximity to the cross-flow channel. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). The magnetic resin bead concentration depends on the desired binding capacity, and the desired solution viscosity, and the magnetic resin bead size depends on the appropriate fluid dynamics for equilibrium and affinity interactions, e.g., solution viscosity dependence and surface ligand density dependence, respectively. depends on the surface area to volume ratio that enables In embodiments, the diameter of the magnetic resin beads is submicron to micron.

Material selection of the affinity-based fluid purification module is important for robust stability and negligible leachate to enable recycling and reuse of the affinity-based fluid purification module. Magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

The agitation capacity and at least one low pH equilibrium vessel volume of the affinity-based fluid purification module enables input flow rate and throughput, equilibration time to enable debinding kinetics, and dilution during equilibration time to reach 1X final buffer salt concentration. Consider a 10X low pH elution buffer. Low pH elution buffers depend on the binding affinity of the desired biological product (e.g., monoclonal antibody) to the magnetic resin bead surface ligand, which changes include pH, ionic strength, or the use of organic and/or inorganic salts. can be included

The throughput of the affinity-based fluid purification module can be increased by multiplexing several fluid elements or chips in series or parallel. Additionally, several fluidic elements or chips connected in series or parallel may be required to enable complete purification. Additionally, the regenerative equilibrium vessel maintains the correct concentration of magnetic resin beads and may require a permanent magnetic field and waste line for effective recycling. Virus inactivation and removal can be achieved during low pH elution buffer equilibration and subsequent fluid handling steps, depending on the buffer composition and pH.

The affinity-based fluid purification module may include at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange fractions comprising biological products.

The affinity-based fluid purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (eg, flow sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the affinity-based fluid purification module uses a mobile affinity resin that can be regenerated and recycled in situ to more efficiently process the resin. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Positive Charge Based Fluid Purification Module

As provided herein, a positive charge based fluid purification module is described for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation. The positive charge based fluid purification module also includes a suspension of cationic magnetic resin beads, wherein the surface of the magnetic resin beads selectively associates with the biological product based on charge or electrostatic interaction at a specific pH and ionic strength. It includes a cationic functional group configured to.

The positive charge-based fluid purification module includes at least one associative equilibrium vessel that allows charge or electrostatic association of a biological product with a surface of a magnetic resin bead; at least one dissociation equilibrium vessel allowing dissociation of the biological product from the magnetic resin bead surface; At least one magnetic field and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to manipulate the flow path of the magnetic resin beads in the heterogeneous mixture, and fire the magnetic resin beads coupled to the biological product. at least one first hybrid cross-flow fluid element or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any of these) comprising a cross-flow channel for separation from small impurities in the homogeneous mixture any combination) (FIG. 28); at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to manipulate the flow path of the magnetic resin beads from the biological product, and separating the magnetic resin beads from the biological product. at least one second hybrid cross-flow fluid element or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any combination thereof) comprising a cross-flow channel for ; at least two buffer systems; at least one magnetic resin bead regeneration buffer system; at least one regeneration equilibration vessel configured to enable recycling of the magnetic resin beads; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the positive charge based fluid purification module (FIG. 30) enables continuous biomagnetic purification and provides a small footprint.

The association equilibrium vessel volume and agitation capacity of the at least one positive charge based fluid purification module takes into account association kinetics, magnetic resin bead concentration, and input flow rate and throughput enabling charge or electrostatic association capacity, equilibration time and agitation rate. . The association buffer for the positive charge based fluid purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Considerations for such buffers specifically include pH, ionic strength, use of surfactants, or use of organic and/or inorganic salts to maintain favorable charge or electrostatic interactions between the target monoclonal antibody and the positively charged bead surface. included

The cross-flow channel size of the positive charge-based fluid purification module depends on the solution viscosity and magnetic resin bead concentration, and the input flow rate and throughput volume. Piezoelectric or acoustic actuators allow for desired magnetic resin bead deflection or manipulation of the magnetic resin bead flow path, and a physical position and energy (eg, frequency) that enables a type of piezoelectric crystal. Dielectrophoretic electrodes take into account the optional type and design that enables the desired magnetic resin bead deflection, the number and spacing of electrodes that allow the desired bead deflection, the applied voltage that allows the desired bead deflection, and the electrode material.

The magnetic field strength and field effect depend on the flow rate and magnetic resin bead concentration, size, saturation magnetization and susceptibility, and/or proximity to the cross-flow channel. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). The magnetic resin bead concentration depends on the desired charge or electrostatic association capacity and the desired solution viscosity, and the magnetic resin bead size depends on the equilibrium and charge or electrostatic interactions, e.g., solution viscosity dependence and surface charge density dependence, respectively. It depends on the surface area to volume ratio required to enable adequate fluid dynamics for action. In embodiments, the diameter of the magnetic resin beads is submicron to micron.

Material selection of the positive charge-based fluid purification module is important for robust stability and negligible leachate to enable recycling and reuse in the positive charge-based fluid purification module.

The choice of cationic surfaces for positive charge based fluid purification modules is important and can include cationic polymers, net positively charged peptides or proteins, and amine functionalities, the choice being positively charged beads within a defined buffer (pH and ionic strength). It is based on achieving an appropriate charge or electrostatic interaction and association stability between the surface and the biological product.

The volume of the dissociation equilibrium vessel and the agitation capacity of at least one of the positive charge based fluid purification module is determined by the input flow rate and throughput, the equilibration time to enable dissociation kinetics, and the equilibration time to allow dilution during the equilibration time to reach the 1X final buffer salt concentration of 10X. Consider a dissociation buffer. The dissociation buffer depends on the strength of the charge, or the electrostatic interaction between the desired biological product (e.g., monoclonal antibody) and the cationic surface of the magnetic resin bead, changes include pH, ionic strength, use of surfactants, or the use of organic and/or inorganic salts. In embodiments, several dissociation equilibrium vessels containing separate buffers of different pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

The throughput of a positive charge-based fluid purification module can be increased by multiplexing several fluid elements or chips in series or parallel. Additionally, several fluidic elements or chips connected in series or parallel may be required to enable complete purification. In addition, the regeneration equilibration vessel maintains the correct concentration of magnetic resin beads, and may require a permanent magnetic field and waste line for effective recycling. Virus inactivation and removal depends on buffer composition and pH, associative or dissociative buffer equilibration and subsequent fluid handling. can be achieved during this phase.

The positive charge based fluid purification module may include at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange a fraction comprising biological products.

The positive charge based fluid purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (eg, flow sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the positive charge-based fluid purification module uses a mobile affinity resin that can be regenerated and recycled in situ, enabling more efficient use of the resin. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Negative Charge Based Fluid Purification Module

As provided herein, a negative charge based fluid purification module is described for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation. In addition, the negative charge based fluid purification module includes a suspension of anionic magnetic resin beads, wherein the surface of the magnetic resin beads selectively associates with the biological product based on charge or electrostatic interaction at a specific pH and ionic strength. It includes an anionic functional group configured to.

The negative charge based fluid purification module includes at least one associative equilibrium vessel allowing electrical or electrostatic association of a biological product with a surface of a magnetic resin bead; at least one dissociation equilibrium vessel allowing dissociation of the biological product from the magnetic resin bead surface; At least one magnetic field and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to manipulate the flow path of the magnetic resin beads in the heterogeneous mixture, and fire the magnetic resin beads coupled to the biological product. at least one first hybrid cross-flow fluid element or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any of these) comprising a cross-flow channel for separation from small impurities in the homogeneous mixture any combination) (FIG. 28); at least one magnetic field and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to manipulate the flow path of the magnetic resin beads from the biological product, and separating the magnetic resin beads from the biological product. at least one second hybrid cross-flow fluid element or chip (e.g., microfluidic, mesofluidic, millifluidic, macrofluidic element or chip, or any combination thereof) comprising a cross-flow channel for ; at least two buffer systems; at least one magnetic resin bead regeneration buffer system; at least one regeneration equilibration vessel configured to enable recycling of the magnetic resin beads; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the negative charge based fluid purification module (FIG. 30) enables continuous biomagnetic purification and provides a small footprint.

The association equilibrium vessel volume and agitation capacity of at least one of the negative charge-based fluid purification module takes into account association kinetics, magnetic resin bead concentration, and input flow rate and throughput enabling charge or electrostatic association capacity, equilibration time, and agitation rate. do. Buffers for negative charge based fluid purification modules depend on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Considerations for such buffers specifically include pH, ionic strength, use of surfactants, or use of organic and/or inorganic salts to maintain a favorable charge or electrostatic interaction between the target monoclonal antibody and the negatively charged bead surface. included

The cross-flow channel size of the negative charge-based fluid purification module depends on the solution viscosity and magnetic resin bead concentration, and the input flow rate and throughput volume. Piezoelectric or acoustic actuators allow for desired magnetic resin bead deflection or manipulation of the magnetic resin bead flow path, and a physical position and energy (eg, frequency) that enables a type of piezoelectric crystal. Dielectrophoretic electrodes take into account the optional type and design that enables the desired magnetic resin bead deflection, the number and spacing of electrodes that allow the desired bead deflection, the applied voltage that allows the desired bead deflection, and the electrode material.

The magnetic field strength and field effect depend on the flow rate and magnetic resin bead concentration, size, saturation magnetization and susceptibility, and/or proximity to the cross-flow channel. In embodiments, the magnetic field strength ranges from about 0.01 Tesla to about 1 Tesla (eg, up to about 1 Tesla). The magnetic resin bead concentration depends on the desired charge or electrostatic association capacity and the desired solution viscosity, and the magnetic resin bead size depends on the equilibrium and charge or electrostatic interactions, e.g., solution viscosity dependence and surface charge density dependence, respectively. It depends on the surface area to volume ratio required to enable adequate fluid dynamics for action. In embodiments, the diameter of the magnetic resin beads is submicron to micron.

Material selection of the negative charge-based fluid purification module is important for robust stability and negligible leachate to enable recycling and reuse in the negative charge-based fluid purification module.

The selection of anionic surfaces for negative charge-based fluid purification modules is important and can include anionic polymers, net negatively charged peptides or proteins, and carboxyl functional groups, the choice being negatively charged beads in a defined buffer (pH and ionic strength). It is based on achieving an appropriate charge or electrostatic interaction and association stability between the surface and the biological product.

The volume of the dissociation equilibrium vessel and the agitation capacity of at least one of the negative charge based fluid purification module is determined by the input flow rate and throughput, the equilibration time to enable dissociation kinetics, and the equilibration time to allow dilution during the equilibration time to reach the 1X final buffer salt concentration of 10X. Consider a dissociation buffer. The dissociation buffer depends on the strength of the charge, or the electrostatic interaction between the desired biological product (e.g., monoclonal antibody) and the anionic surface of the magnetic resin bead, which changes include pH, ionic strength, use of surfactants, or the use of organic and/or inorganic salts. In embodiments, several dissociation equilibrium vessels containing separate buffers of different pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

The throughput of the negative charge based fluid purification module can be increased by multiplexing several fluid elements or chips in series or parallel. Additionally, several fluidic elements or chips connected in series or parallel may be required to enable complete purification. In addition, the regeneration equilibration vessel maintains the correct concentration of magnetic resin beads, and may require a permanent magnetic field and waste line for effective recycling. Virus inactivation and removal depends on buffer composition and pH, associative or dissociative buffer equilibration and subsequent fluid handling. can be achieved during this phase.

The negative charge based fluid purification module may include at least one tangential flow filtration system operating in a fed-batch or per-through mode to concentrate and buffer exchange a fraction comprising biological products.

The negative charge based fluid purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (eg, flow sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column switching chromatography methods commonly used in the art, the negative charge-based magnetic purification module uses a mobile affinity resin that can be regenerated and recycled in situ and uses the resin more efficiently. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Affinity-based TFF purification module

As provided herein, an affinity-based TFF purification module is described for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is maintained during steady state operation. consistent and consistent The affinity-based TFF purification module also includes a suspension of affinity resin beads, wherein the surface of the resin beads is, without limitation, configured to bind selectively to the biological product: protein A, protein G, protein L, It is linked to an antigenic protein, protein, receptor, antibody, or aptamer.

The affinity-based TFF purification module comprises at least one equilibration vessel allowing binding of a biological product to the resin bead surface, and a hollow fiber membrane filter for separating the resin beads bound to the biological product from minor impurities in the heterogeneous mixture. at least one first tangential flow filtration system comprising; at least one second tangential flow filtration comprising at least one low pH equilibrated vessel enabling debinding of the biological product from the resin bead surface, and a hollow fiber membrane filter for separating the resin beads from the unbound biological product. system; at least one third tangential flow filtration system comprising at least one regeneration equilibrium vessel and a hollow fiber membrane filter for concentrating and buffer exchanging the resin beads to allow for their recycling and reuse; at least one collection vessel and at least one fourth tangential flow filtration system enabling concentration and buffer exchange of the biological product; at least two buffer systems; at least one resin bead recycling buffer system; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the affinity-based TFF purification module (FIG. 31) enables continuous biological purification and provides a small footprint.

The equilibrium vessel volume and agitation capacity of the at least one affinity-based TFF purification module takes into account binding kinetics, resin bead concentration, and input flow rate and throughput enabling the binding capacity, equilibration time, and agitation rate. Equilibration buffers for affinity-based TFF purification modules depend on the desired biological product (eg, monoclonal antibody) and the minor impurities to be removed. Considerations for the buffer include pH, ionic strength, use of surfactants, or use of organic and/or inorganic salts.

The hollow fiber membrane filter length and surface area of the affinity-based TFF purification module depend on solution viscosity, small impurity and resin bead concentrations, and input flow rate and throughput volume. Although hollow fiber membrane materials include PES, mPES, or MCE. It is selected from low protein binding materials that are not limited thereto. The pore size of the hollow fiber membrane is selected from the range of about 10 kDa to about 1 μm. The inner diameter of the hollow fiber membrane is selected from the range of about 0.5 mm to about 5 mm.

The concentration of the resin beads depends on the desired binding capacity, and the desired solution viscosity, and the size of the resin beads, for example, the solution viscosity dependence and the surface ligand density dependence, respectively, for the equilibrium and the appropriate hydrodynamics for the affinity interaction. depends on the surface area to volume ratio that enables In embodiments, the diameter of the resin bead is microns.

Resin bead selection for affinity-based TFF purification modules is important for robust stability and negligible leachables to enable recycling and reuse of affinity-based purification modules.

The agitation capacity and volume of the at least one low pH equilibration vessel of the affinity-based TFF purification module depends on the input flow rate and throughput, the equilibration time to enable debinding kinetics, and the low pH elution buffer, e.g., 1X final buffer salt concentration. Consider a 10X low pH elution buffer to allow dilution during the equilibration time to reach. Low pH elution buffers depend on the binding affinity of the desired biological product (e.g., monoclonal antibody) to the resin bead surface ligand, and changes may include pH, ionic strength, or the use of organic and/or inorganic salts. can

At least one regeneration equilibration vessel of the affinity-based TFF purification module includes at least one third tangent to allow concentration and buffer exchange of the resin beads to return the resin beads to their initial state to enable recycling and reuse of the resin beads. Used with flow filtration systems.

At least one collection vessel of the affinity-based TFF purification module is used with at least one fourth tangential flow filtration system enabling concentration and buffer exchange of fractions comprising biological products.

In embodiments, the at least one equilibration vessel, the at least one low pH equilibration vessel, and the at least one regeneration equilibration vessel of an affinity-based TFF purification module provide continuous flow of filtrate through at least one additional vessel in a parallel flow path. It can include a single vessel that is switched between corresponding tangential flow filtration systems to enable purification and regeneration of the resin beads using appropriate buffers while maintaining

In embodiments, regeneration of the resin beads comprises at least one low pH equilibrating vessel and an affinity-based TFF purification module configured to include both a low pH elution buffer and a regeneration buffer to enable purification, concentration and buffer exchange. This can be achieved by using at least one second tangential flow filtration system to regenerate the resin beads without the need for a separate regeneration equilibration vessel and corresponding tangential flow filtration system.

Virus inactivation and/or removal can be achieved during low pH elution buffer equilibration and subsequent fluid handling steps, depending on the buffer composition and pH.

The affinity-based TFF purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (eg, flow sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the affinity-based TFF purification module uses a mobile affinity resin that can be regenerated and recycled in situ, making resin more efficient. and enables continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Positive charge-based TFF purification module

As provided herein, a positive charge based TFF purification module is described for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is consistent during steady state operation. and become constant The positive charge based TFF purification module also includes a suspension of cationic resin beads, wherein the surface of the resin beads is configured to selectively associate with the biological product based on charge or electrostatic interaction at a specific pH and ionic strength. contains sexual functional groups.

The positive charge based TFF purification module includes at least one association equilibrium vessel enabling charge or electrostatic association of the biological product with the surface of the resin beads, and hollow fibers for separating the resin beads associated with the biological product from small impurities in the heterogeneous mixture. at least one first tangential flow filtration system comprising a membrane filter; at least one second tangential flow filtration system comprising at least one dissociation equilibrium vessel enabling dissociation of the biological product from the resin bead surface, and a hollow fiber membrane filter for separating the resin beads from the dissociated biological product; at least one third tangential flow filtration system comprising at least one regeneration equilibrium vessel and a hollow fiber membrane filter for concentrating and buffer exchanging the resin beads to allow for their recycling and reuse; at least one collection vessel and at least one fourth tangential flow filtration system enabling concentration and buffer exchange of the biological product; at least two buffer systems; at least one resin bead recycling buffer system; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the positive charge-based TFF purification module (FIG. 32) enables continuous biological purification and provides a small footprint.

The association equilibrium vessel volume and agitation capacity of the at least one positive charge-based TFF purification module takes into account association kinetics, resin bead concentration, and input flow rate and throughput enabling charge or electrostatic association capacity, equilibration time, and agitation rate. The association buffer for the positive charge based TFF purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Considerations for such buffers specifically include pH, ionic strength, use of surfactants, or use of organic and/or inorganic salts to maintain favorable charge or electrostatic interactions between the target monoclonal antibody and the positively charged bead surface. included

The hollow fiber membrane filter length and surface area of the positive charge-based TFF purification module depend on solution viscosity, small impurity and resin bead concentrations, and input flow rate and throughput volume. Although hollow fiber membrane materials include PES, mPES, or MCE. It is selected from low protein binding materials that are not limited thereto. The pore size of the hollow fiber membrane is selected from the range of about 10 kDa to about 1 μm. The inner diameter of the hollow fiber membrane is selected from the range of about 0.5 mm to about 5 mm.

Resin bead selection for positive charge-based TFF purification modules is important for robust stability and negligible leachate to enable recycling and reuse in positive charge-based TFF purification modules.

The choice of cationic surfaces for positive charge-based TFF purification modules is important and can include cationic polymers, net positively charged peptides or proteins, and amine functional groups, the choice being positively charged bead surfaces within a defined buffer (pH and ionic strength). It is based on achieving the appropriate charge or electrostatic interaction and association stability between the biological product and the biological product.

The volume and agitation capacity of at least one dissociation equilibrium vessel of the positive charge-based TFF purification module is determined by the input flow rate and throughput, the equilibration time to enable dissociation kinetics, and the equilibration time to reach the dissociation buffer, e.g., 1X final buffer salt concentration. Consider a dissociation buffer of 10X to allow for dilution. The dissociation buffer depends on the strength of the charge, or electrostatic interaction between the desired biological product (e.g., monoclonal antibody) and the resin bead cationic surface, which changes include pH, ionic strength, use of surfactants, or The use of organic and/or inorganic salts may be included. In some aspects, multiple dissociation equilibrium vessels are used with multiple tangential flow filtration systems to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

At least one regeneration equilibrium vessel of the positive charge-based TFF purification module includes at least one third tangential flow that allows concentration and buffer exchange of the resin beads to return the resin beads to their initial state to enable recycling and reuse of the resin beads. Used with filtration systems.

The at least one collection vessel of the positive charge based TFF purification module is used with at least one fourth tangential flow filtration system enabling concentration and buffer exchange of the fraction comprising biological products.

In embodiments, the at least one association equilibrium vessel, the at least one dissociation equilibrium vessel, and the at least one regeneration equilibrium vessel of the positive charge-based TFF purification module provide a continuous flow of filtrate through at least one additional vessel in a parallel flow path. It may include a single vessel that is switched between corresponding tangential flow filtration systems to enable purification and regeneration of the resin beads using appropriate buffers while maintaining the same.

In embodiments, the regeneration of the resin beads comprises at least one second tangential flow filtration system of a positive charge based TFF purification module configured to include both a low pH elution buffer and a regeneration buffer to enable purification, concentration and buffer exchange; and by using only at least one dissociation equilibrium vessel to regenerate the resin beads without the need for a separate regeneration equilibrium vessel and corresponding tangential flow filtration system.

Virus inactivation and/or removal may be achieved during association or dissociation buffer equilibration and subsequent fluid handling steps, depending on the buffer composition and pH.

The positive charge-based TFF purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (eg, flow sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the positive charge-based TFF purification module uses a mobile affinity resin that can be regenerated and recycled in situ, enabling more efficient use of the resin. , enabling continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Negative charge-based TFF purification module

As provided herein, negative charge based TFF purification modules are described for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is consistent during steady state operation. and become constant The negative charge based TFF purification module also includes a suspension of anionic resin beads, wherein the surface of the resin beads is an anion configured to selectively associate with the biological product based on charge or electrostatic interaction at a specific pH and ionic strength. contains sexual functional groups.

The negative charge based TFF purification module includes at least one association equilibrium vessel enabling charge or electrostatic association of the biological product with the surface of the resin beads, and a hollow fiber for separating the resin beads associated with the biological product from small impurities in the heterogeneous mixture. at least one first tangential flow filtration system comprising a membrane filter; at least one second tangential flow filtration system comprising at least one dissociation equilibrium vessel enabling dissociation of the biological product from the resin bead surface, and a hollow fiber membrane filter for separating the resin beads from the dissociated biological product; at least one third tangential flow filtration system comprising at least one regeneration equilibrium vessel and a hollow fiber membrane filter for concentrating and buffer exchanging the resin beads to allow for their recycling and reuse; at least one collection vessel and at least one fourth tangential flow filtration system enabling concentration and buffer exchange of the biological product; at least two buffer systems; at least one resin bead recycling buffer system; at least one sensor or detector; and at least one fluid handling pump.

The equipment design for the negative charge-based TFF purification module (FIG. 32) enables continuous biological purification and provides a small footprint.

The association equilibrium vessel volume and agitation capacity of the at least one negative charge-based TFF purification module takes into account association kinetics, resin bead concentration, and input flow rate and throughput enabling charge or electrostatic association capacity, equilibration time, and agitation rate. The association buffer for the negative charge based TFF purification module depends on the desired biological product (eg monoclonal antibody) and the small impurities to be removed. Considerations for such buffers specifically include pH, ionic strength, use of surfactants, or use of organic and/or inorganic salts to maintain a favorable charge or electrostatic interaction between the target monoclonal antibody and the negatively charged bead surface. included

The hollow fiber membrane filter length and surface area of the negative charge-based TFF purification module depend on solution viscosity, small impurity and resin bead concentrations, and input flow rate and throughput volume. Although hollow fiber membrane materials include PES, mPES, or MCE. It is selected from low protein binding materials that are not limited thereto. The pore size of the hollow fiber membrane is selected from the range of about 10 kDa to about 1 μm. The inner diameter of the hollow fiber membrane is selected from the range of about 0.5 mm to about 5 mm.

Resin bead selection for negative charge-based TFF purification modules is important for robust stability and negligible leachables to enable recycling and reuse in negative charge-based purification modules.

The selection of anionic surfaces for negative charge-based TFF purification modules is important and can include anionic polymers, net negatively charged peptides or proteins, and carboxyl functional groups, the selection being made on negatively charged bead surfaces in defined buffers (pH and ionic strength). It is based on achieving the appropriate charge or electrostatic interaction and association stability between the biological product and the biological product.

The volume and agitation capacity of at least one dissociation equilibrium vessel of the negative charge based TFF purification module is determined by the input flow rate and throughput, the equilibration time to enable the dissociation kinetics, and the equilibration time to reach the dissociation buffer, e.g., 1X final buffer salt concentration. Consider a dissociation buffer of 10X to allow for dilution. The dissociation buffer depends on the strength of the charge, or electrostatic interaction between the desired biological product (e.g., monoclonal antibody) and the resin bead anionic surface, which changes include pH, ionic strength, use of surfactants, or The use of organic and/or inorganic salts may be included. In some aspects, multiple dissociation equilibrium vessels are used with multiple tangential flow filtration systems to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

At least one regeneration equilibrium vessel of the negative charge-based TFF purification module includes at least one third tangential flow that allows concentration and buffer exchange of the resin beads to return the resin beads to their initial state, enabling recycling and reuse of the resin beads. Used with filtration systems.

At least one collection vessel of the negative charge based TFF purification module is used with at least one fourth tangential flow filtration system enabling concentration and buffer exchange of the fraction comprising biological products.

In embodiments, the at least one association equilibrium vessel, the at least one dissociation equilibrium vessel, and the at least one regeneration equilibrium vessel of the negative charge based TFF purification module provide continuous flow of filtrate through at least one additional vessel in a parallel flow path. It may include a single vessel that is switched between corresponding tangential flow filtration systems to enable purification and regeneration of the resin beads using appropriate buffers while maintaining the same.

In embodiments, the regeneration of the resin beads comprises at least one second tangential flow filtration system of a negative charge based TFF purification module configured to include both a low pH elution buffer and a regeneration buffer to enable purification, concentration and buffer exchange; and by using only at least one dissociation equilibrium vessel to regenerate the resin beads without the need for a separate regeneration equilibrium vessel and corresponding tangential flow filtration system.

Virus inactivation and/or removal may be achieved during association or dissociation buffer equilibration and subsequent fluid handling steps, depending on the buffer composition and pH.

The negative charge-based TFF purification module may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (eg, flow sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process.

Unlike the conventional packed column chromatography and column shift chromatography methods commonly used in the art, the negative charge-based TFF purification module uses a mobile affinity resin that can be regenerated and recycled in situ, enabling more efficient use of the resin. , enabling continuous processing in a small footprint without the concerns of traditional column capacity limitations.

Isoelectric point based fluid purification module

As provided herein, an isoelectric point based fluid purification module for separating a mixture into two or more fractions, a fraction containing at least one biological product, is described. The isoelectric point-based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is, for example, , can be consistent and constant during steady state operation.

In embodiments, an isoelectric point-based fluid purification module comprises a fluid channel created between two parallel plates; electric field or electric field gradient perpendicular to the direction of fluid flow; a fluidic device (eg, mesofluidic, millifluidic, macrofluidic device, or any combination thereof) having an aqueous ionic solution; (FIG. 33); at least one bubble removal or degassing system for removing electrolytic bubbles near the point of generation by a vacuum system to create a bubble-free main separation channel and to enable continuous and long-term operation; at least one liquid circuit breaker; at least one buffer or amphoteric electrolyte system; at least one electrode solution; at least one sensor or detector; at least one fluid handling pump; and at least one free flow electrophoresis device comprising at least one collection vessel.

In another embodiment, an isoelectric based fluid purification module includes a fluid channel created between two parallel plates; electric field or electric field gradient perpendicular to the direction of fluid flow; at least one first free flow electrophoretic device comprising a fluidic element (eg, mesofluidic, millifluidic, macrofluidic element, or any combination thereof) having a; and a fluid channel created between the two parallel plates; electric field or electric field gradient perpendicular to the direction of fluid flow; and an aqueous ionic solution; In this case, each free flow electrophoresis device is connected in series and can operate in an independent mode of operation allowing purification (FIGS. 34 to 36). For example, without intending to be limiting, at least one first free flow electrophoretic device can operate in an isoelectric focusing mode and at least one second free flow electrophoretic device can operate in an isokinetic mode to improve separation resolution. can be raised

In embodiments, an isoelectric point-based fluid purification module comprises a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across the main separating channel (e.g. , at least one first free flow electrophoretic device comprising a fluidic element (eg, a mesofluidic, millifluidic, macrofluidic element, or any combination thereof) having a pH ranging from about 2 to about 10; and a fluid channel created between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a slight pH gradient (e.g., in the pH range of about 5 to about 8) across the main separating channel. a fluidic element (eg, mesofluidic, millifluidic, macrofluidic element, or any combination thereof) comprising at least one second free flow electrophoretic device; at least one degassing or degassing system; at least one liquid circuit breaker; at least one buffer or amphoteric electrolyte system; at least one electrode solution; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump (FIG. 34). For example, at least one first free-flow electrophoretic device and at least one second free-flow electrophoretic device are connected in series and operate in an isoelectric focusing mode.

In embodiments, an isoelectric point-based fluid purification module comprises a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant basic pH across a main separation channel without a pH gradient ( at least one first free flow electrophoretic device comprising a fluidic element (eg, a mesofluidic, millifluidic, macrofluidic element, or any combination thereof) having, for example, a pH greater than 7; and a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (e.g., pH less than 7) across the main separating channel with no pH gradient. at least one second free flow electrophoretic device comprising a fluidic element (eg, a mesofluidic, microfluidic, macrofluidic element, or any combination thereof); at least one degassing or degassing system; at least one liquid circuit breaker; at least one buffer or amphoteric electrolyte system; at least one electrode solution; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump (FIG. 35). For example, at least one first free-flow electrophoretic device and at least one second free-flow electrophoretic device are connected in series and operate in a zone electrophoresis mode.

In embodiments, an isoelectric based fluid purification module comprises a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient across the main separating channel (e.g., about at least one first free flow electrophoretic device comprising a fluidic element (eg, a mesofluidic, millifluidic, macrofluidic element, or any combination thereof) having a pH range of 4 to about 9; and a fluidic channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution). at least one second free flow electrophoretic device comprising a fluidic element (eg, a mesofluidic, millifluidic, macrofluidic element, or any combination thereof) comprising: at least one degassing or degassing system; at least one liquid circuit breaker; at least one buffer or amphoteric electrolyte system; at least one electrode solution; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump (FIG. 36). For example, at least one first free-flow electrophoresis device and at least one second free-flow electrophoresis device are connected in series and operate in an isoelectric focusing mode and an isokinetic electrophoresis mode, respectively.

Alternatively, the isoelectric point-based fluid purification module includes at least one first fluid element (eg, mesofluid, millifluid) comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a prescribed unidirectional force. , a macrofluidic device, or any combination thereof); A fluid channel created between two parallel plates, a free flow with an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient (e.g., pH range from about 4 to about 9) across the main separating channel. at least one second fluid element comprising an electrophoretic device; and a fluidic channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution). at least one third fluidic element comprising a free flow electrophoresis device having; at least one degassing or degassing system; at least one liquid circuit breaker; at least one buffer or amphoteric electrolyte system; at least one electrode solution; at least one collection vessel; at least one sensor or detector; and at least one fluid handling pump (FIG. 37). For example, at least one first fluidic element having at least one optional dielectrophoretic electrode, at least one second fluidic element comprising a free flow electrophoretic device, and at least one fluidic element comprising a free flow electrophoretic device. The third fluid element is connected in series and operates in such a way that the mixture containing the biological product is pre-classified prior to purification through an isoelectric focusing mode and an isokinetic electrophoresis mode by the second and third fluid elements or chips, respectively. .

In embodiments, a fluid channel created between two parallel plates to increase separation resolution, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fluid element having an aqueous ionic solution (e.g., mesofluid, millifluid) Additional subsequent free flow electrophoretic devices including fluids, macrofluidic devices, or any combination thereof may be used. For example, without intending to be limiting, an amphoteric electrolyte solution that can create a refined pH gradient (e.g., a pH range of about 7.1 to about 7.6) across the main separation channel to increase the separation resolution of monoclonal antibodies. Additional free flow electrophoresis devices with

The equipment design for the isoelectric point based fluid purification module (FIGS. 33-37) enables continuous biological purification and provides a small footprint.

In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (eg, a platinum wire electrode) to function as an anode or cathode.

In embodiments, an isoelectric point based fluid purification device includes at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper functioning in contact with an anode or cathode, for example sulfuric acid and sodium hydroxide, respectively. In other embodiments, at least one electrode solution is present in a main isolation channel configured to enable proper functioning of the anode or cathode, such as Tris buffered saline, flowing through the main isolation channel, the anode channel, and the cathode channel. It includes the same amphoteric electrolyte composition as described above.

In embodiments, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O ₂ and H ₂ gas bubbles generated in the electrode channel under an applied voltage through a vacuum system (FIG. 38 ). In some embodiments, the removal of electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. The removal of electrolysis bubbles directly from the electrode channels creates a main bubble-free isolation channel. For example, a bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over an electrode channel that can continuously remove electrolytic bubbles at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -0.05 bar to about -0.4 bar.

Fluidic channel dimensions and voltages applied to the channels to create orthogonal electric fields or electric field gradients that allow for protein separation at high flow rates (eg, 1 mL/min or higher) can be used to determine the thermal resistance of biological products (eg, monoclonal antibodies) (eg, monoclonal antibodies). Implementation of a powerful active cooling system or heat sink to dissipate Joule heat and maintain a desired operating temperature, e.g., from about 4°C to about 50°C, preferably from 4°C to about 37°C, to ensure thermal stability. may require For example, an active cooling system may include an aluminum thermal chuck that includes a cooled circulating water/propylene glycol jacket with a feedback control loop to maintain a constant temperature in the range of about 10° C. to about 25° C. can In addition, the dimension of the channel is an important parameter enabling proper protein separation, and varies depending on the number of proteins to be separated and the range of isoelectric points of each protein.

The back pressure within an isoelectric point-based fluid purification device depends on channel shape and dimensions, inlet and outlet openings and/or tubing diameters, and input flow rate. For example, the back pressure ranges from about 0.5 psi to about 10 psi. In some examples, the back pressure is controlled by, for example, without intending to limit, a needle valve.

For example, to perform in-line sensing and detection using a flow sensor, temperature sensor, conductivity sensor, pH sensor, refractive index detector, UV detector, back pressure sensor, or any combination thereof, the solution must be a voltage-free solution. In embodiments, an isoelectric point-based fluid purification module includes at least one liquid circuit breaker or disconnects downstream of a fluid element and upstream of at least one in-line sensor or detector to perform sensing or detection in a voltage-free solution. It should be possible (Fig. 39).

The throughput of the isoelectric point based fluid purification module can be increased by multiplexing several free flow electrophoresis devices in series or parallel. Additionally, several fluidic elements or chips connected in series or parallel may be required to allow for proper purification. Virus inactivation and removal can be achieved during an isoelectric point based fluid treatment step.

An isoelectric based fluid purification module is contemplated that may include in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques. In addition, in-line analytical measurement techniques (e.g., flow sensors, temperature sensors, pH sensors, conductivity sensors, pressure sensors, optical density measurement devices, UV detectors, RI detectors) can be used to activate feedback control mechanisms in the process. .

Continuous dynamic filtration approach

The rate of transport of the rolled filter membrane across the membrane support structure may be constant, or a feedback mechanism (e.g., rotary encoder , can be changed corresponding to the traction encoder wheel. Alternatively, the transport of the rolled filter membrane across the membrane support structure may be staged, wherein the vacuum is removed during stepping and then reapplied after stepping. In this mode of operation, at least one output head may have xy rastering or rθ rastering capability, movement along the z-axis, and/or at least one additional dynamic filtering device operating in parallel to maintain continuous operation. can be used The stepping phenomenon can also be achieved by having a pick-and-place robotic system place individual membranes (e.g., circular membrane pieces) onto a membrane support structure, where a vacuum is removed during the pick-and-place mechanics and then reapplied after placement. . In this mode of operation, at least one output head may have xy rastering or rθ rastering capability, movement along the z-axis, and/or at least one additional dynamic filtering device operating in parallel to maintain continuous operation. can be used Also contemplated herein is a mode of operation in which a positive pressure is used to generate a filtrate to induce a heterogeneous mixture across a filter membrane. For example, without intending to be limiting, where a dynamic filtration device includes a pick-and-place robotic system to place the individual membranes, an xy rastering output head moving along the z-axis contacts the membrane surface so that the inhomogeneous mixture displaces the membrane. Can be used to cross. Methods of pre-wetting the filter membrane to increase transport across the membrane are also contemplated herein. The at least one vacuum collection vessel may be used until full and equilibrate with atmospheric pressure, or may be continuously evacuated in vacuum during operation. Additionally, the use of at least one additional dynamic filtration device operated in parallel to enable continuous processing is also contemplated herein. Similarly, a dynamic filtration device may include multiple feed reels of different types (e.g., materials and/or pore sizes) of filter membranes that may be stacked and transported at the same rate across an active target area to enhance filtration. may include

Continuous Loop Conveyor Approach

At least one magnetic field of the continuous loop conveyor device performing the affinity-based magnetic purification and/or charge-based magnetic purification steps may be generated by an electromagnet or a permanent magnet (eg, a neodymium magnet). The magnetic field can be applied as an on/off toggle or permanently on (FIGS. 14, 15, 16, 17). If the magnetic field is generated by an electromagnet, it can be switched on/off by turning the electromagnet on or off. If the magnetic field is generated by permanent magnets, on/off switching can be accomplished by mechanically changing the placement of the magnets from far to near, eg, within 5 mm of the transport vessel wall surface. Alternatively, if the magnetic field is generated by a permanent magnet, on/off switching can be accomplished by moving the container in or out of proximity (eg, within 5 mm) of the magnet. Also, loop conveyor devices may include several distinct magnetic field locations, wherein the distinct magnetic field locations within the devices may additionally require shielding. Additionally, mixing of the magnetic resin beads may be accomplished by placing the at least one transport vessel between two separate opposing magnetic fields that switch between an on state and an off state. Additionally, the use of at least one additional affinity-based magnetic purification or charge-based magnetic purification device connected in parallel to enable continuous processing is also contemplated herein.

A continuous pick-and-place robotic device approach

At least one magnetic field of the continuous pick-and-place robotic device that performs the affinity-based magnetic purification and/or charge-based magnetic purification steps may be generated by an electromagnet or a permanent magnet (eg, a neodymium magnet). The magnetic field can be applied with an on/off toggle or permanently on (FIGS. 18 and 19). If the magnetic field is generated by an electromagnet, it can be switched on/off by turning the electromagnet on or off. If the magnetic field is generated by permanent magnets, on/off switching can be accomplished by mechanically changing the placement of the magnets from far to near, eg, within 5 mm of the transport vessel wall surface. Alternatively, if the magnetic field is generated by a permanent magnet, on/off switching can be accomplished by moving the container in or out of proximity (eg, within 5 mm) of the magnet. Also, loop conveyor devices may include several distinct magnetic field locations, wherein the distinct magnetic field locations within the devices may additionally require shielding. Additionally, mixing of the magnetic resin beads may be accomplished by placing the at least one transport vessel between two separate opposing magnetic fields that switch between an on state and an off state. Additionally, the use of at least one additional affinity-based magnetic purification or charge-based magnetic purification device connected in parallel to enable continuous processing is also contemplated herein.

Continuous Mechanical Rotation System Device Approach

The seal between the container assembly and at least one gasket lid of a continuous mechanical rotating device that performs affinity-based purification and/or charge-based purification step systems is a mechanism designed to compress the gasket and secure the lid into a sealed three-dimensional shape. It can be formed by (Figs. 21-23). In embodiments, the mechanism used to form the seal is a clamping system or interlocking system. In some embodiments, the mechanism used to form the seal is a screw cap system. In another embodiment, the seal is formed by mechanically pressing the lid down evenly over the container from above with a motorized system. In aspects, a mechanical system or a robotic system may be used to enable the seal to be reproducibly formed and broken in a reversible and repeatable manner over several cycles to enable continuous operation. For example, the integrity of the seal can be analyzed using a leak test after pressurization. Additionally, the buffer inlets in at least one lead may be arranged to create a circular or vortex-like mixed flow pattern. Additionally, the use of at least one additional affinity-based purification or charge-based purification device connected in parallel to enable continuous processing is contemplated herein.

Continuous step-by-step linear system device approach

The vessels of the staged linear system are configured to receive a continuous input flow. Combine, wash, elution, and regeneration (for affinity-based purification) and association, wash, dissociation, and regeneration (for charge-based purification) process steps are performed by connecting each vessel to a manifold that allows automated liquid handling. It becomes (Figs. 24 to 25). In embodiments, each vessel of the staged linear system can be configured to perform different process steps simultaneously to enable continuous operation. For example, without intending to be limiting, one vessel may perform a combining process step while another vessel performs an elution process step.

Continuous Hybrid Fluid Device Approach

The magnetic field of at least one of the continuous hybrid fluid elements (FIG. 28) performing the affinity-based fluid purification and/or charge-based fluid purification steps may be an electromagnet, permanent magnet (eg, neodymium magnet), or a patterned magnet. can be created by Independent permanent magnets may require local shielding. Additionally, a combination of magnetic and piezoelectric components, or a combination of magnetic and dielectrophoretic components, may require precise placement to achieve high flow rates. Additional cross-flow channel designs are considered. Alternatively, a T-junction channel design is also contemplated. It is also a coarse/fine screen approach for selecting biological products of interest (eg, monoclonal antibodies) based on isoelectric point. Also, multiple elution or dissociation is contemplated. The equilibrium vessel may be a batch, fed-batch, continuous feed and discharge vessel, or a plug flow reactor with appropriate residence time. Additionally, multiple fluidic devices or chips are multiplexed in series or parallel to increase the throughput of the affinity-based fluid purification or charge-based fluid purification module. Also contemplated herein is the use of at least one additional affinity-based fluid purification or charge-based fluid purification device connected in parallel to enable continuous processing.

Continuous tangential flow filtration approach

The equilibrium vessels of the affinity-based TFF purification and charge-based TFF purification modules (FIGS. 31-32) can be batch, fed-batch, continuous feed and discharge vessels, or plug flow reactors with appropriate residence times. The use of additional tangential flow filtration systems that enable continuous operation, buffer exchange, diafiltration, ultrafiltration/diafiltration, microfiltration/diafiltration, and/or concentration are also contemplated herein.

Continuous free-flow electrophoresis approach

An electric field perpendicular to the direction of fluid flow is present in aqueous ionic solutions and/or pH gradients, e.g., as described herein, in a fluid channel created between two parallel plates, an electric field or electric field perpendicular to the direction of fluid flow It can be used to purify biological products (eg, monoclonal antibodies) in free flow electrophoresis devices comprising gradients, and aqueous ionic solutions and/or pH gradients (FIGS. 33-37). The dimensions of the fluid channel depend on the flow rate, throughput, time to achieve separation, diffuse band broadening, and magnitude of the electric field, and further depend on the ability of the cooling system to dissipate Joule heat. Additionally, the channel design may have multiple inlets to create a pH gradient by delivering one or more buffers or amphoteric electrolyte systems. The applied voltage may vary depending on the flow rate, pH gradient, biological product charge and/or isoelectric point (eg, monoclonal antibody isoelectric point), or the ability of the cooling system to dissipate Joule heat. The various buffers and/or amphoteric electrolyte systems are chosen to control their charge as they depend on the biological product of interest (eg, the monoclonal antibody of interest) and its inherent isoelectric point (pl). Control of pH and ionic strength and use of organic salts, inorganic salts, acids, bases, zwitterions and/or ampholytes in buffer and/or amphoteric electrolyte systems to achieve a continuous pH gradient effect across the channel. It is considered to establish Alternatively, the pH gradient may be stratified through several inlets delivering separate buffer and/or amphoteric electrolyte systems to achieve a desired gradient, eg, a continuous gradient or a stepwise gradient, without intent to limit. . Also, a combination of coarse and fine pH gradients may be required to purify only biological products (eg, monoclonal antibodies). Considerations regarding robust cooling mechanisms (eg Peltier element, thermal chuck with circulating water/propylene glycol jacket) to enable sufficient heat transfer are also considered. Multiplexing of free flow electrophoresis devices can accommodate higher input flow rates, and thus higher throughputs. Also considered is the possibility of further removal of inactivated virus remaining during the isoelectric point based fluid purification step.

Integration of standard semi-continuous industrial downstream processes

To enable turn-key, end-to-end processes (e.g., from bioreactor-based monoclonal antibody production to final form biological product acquisition), standard semi-continuous industry downstream Process equipment (and steps) can be added to the processes described herein for further purification, buffer exchange, and concentration of purified biological products (eg, monoclonal antibodies) (FIGS. 48 and 49).

Specified virus inactivation and filtration steps may or may not be necessary if virus inactivation and removal is properly performed by the module (eg LRV > 4 for MVM and MuLV viruses). However, the addition of designated viral inactivation and filtration steps is contemplated herein.

Depending on the purity of the biological product (e.g., that of a monoclonal antibody) achieved by the continuous process modules described herein, off-the-shelf tangential flow filtration (TFF), ultrafiltration/diafiltration (UF/ DF), or high-performance tangential flow filtration (HP-TFF) technology run in fed-batch or perfusion mode is a final filter prior to fill-finish operations that may include vial filling, lyophilization, filter sterilization, terminal sterilization, or combinations thereof. It can be used to further purify, buffer exchange, or concentrate biological products. Also contemplated are in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques.

method

Provided herein is a method for continuously purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product at steady state, comprising using the process described herein. As used herein, the terms “steady state” or “dynamic equilibrium” can refer to a system or process that remains stable over time with or without perturbations. For example, an expression host cell, such as a mammalian cell (eg, a CHO cell) or a bacterial cell (eg, E. coli ), can grow to a physiological steady state under constant environmental conditions. A bioreactor is provided that produces the biological product at steady state. In this steady state, growth occurs at a constant specific cell culture growth rate, and all culture parameters remain constant (culture volume, dissolved oxygen concentration, nutrient and product concentrations, pH, cell density, etc.). In addition, environmental conditions may be affected by feedback mechanisms inherent in a bioreactor (e.g., fed-batch, perfusion, or chemostat bioreactor) (e.g., feed/ discharge system). For example, cell density remains constant over time. In another example, the protein concentration is held constant over time.

Modules described herein (e.g., dynamic filtration module, affinity-based magnetic purification module, positive charge-based magnetic purification module, negative charge-based magnetic purification module, affinity-based purification module, positive charge-based purification module, negative charge-based purification module) module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or isoelectric point based fluid purification module A method for continuously purifying a biological product from a heterogeneous mixture derived from a bioreactor that produces the biological product at steady state, comprising utilizing at least one of the purification modules.

In addition, the modules described herein (e.g., dynamic filtration module, affinity-based magnetic purification module, positive charge-based magnetic purification module, negative charge-based magnetic purification module, affinity-based purification module, positive charge-based purification module, negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or isoelectric point A method for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product is also disclosed, comprising using at least one module of a fluid purification module based on the biological product. For example, the modules described herein (e.g., dynamic filtration module, affinity-based magnetic purification module, positive charge-based magnetic purification module, negative charge-based magnetic purification module, affinity-based purification module, positive charge-based purification module) , negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or At least one of the isoelectric point-based fluid purification modules) may be used in place of or in addition to traditional purification techniques used in current batch, single use, or semi-continuous processes known in the art.

Alternatively, the modules described herein (e.g., dynamic filtration module, affinity-based magnetic purification module, positive charge-based magnetic purification module, negative charge-based magnetic purification module, affinity-based purification module, positive charge-based purification module) , negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or A method for purifying a biological product from a mixture not derived from a bioreactor producing the biological product is also disclosed, comprising using at least one module of an isoelectric point based fluid purification module). For example, the modules described herein (e.g., dynamic filtration module, affinity-based magnetic purification module, positive charge-based magnetic purification module, negative charge-based magnetic purification module, affinity-based purification module, positive charge-based purification module) , negative charge based purification module, affinity based fluid purification module, positive charge based fluid purification module, negative charge based fluid purification module, affinity based TFF purification module, positive charge based TFF purification module, negative charge based TFF purification module, and/or At least one of the isoelectric point-based fluid purification modules) may be used in place of or in addition to traditional purification techniques used in current batch, single use, or semi-continuous processes known in the art.

As used herein, the term “module” or “modular” refers to individual distinct parts (e.g., dynamic filtration module, affinity Degree-based magnetic purification module, positive charge-based magnetic purification module, negative charge-based magnetic purification module, affinity-based fluid purification module, positive charge-based fluid purification module, negative charge-based fluid purification module and/or isoelectric point-based fluid purification module module). Additionally, the process may include one or more of the modules described above. The term “modular” refers to and can mean a structure that is composed of a plurality of modular units and can be composed of various structural forms. For example, modular units may be connected together in the form of a transport system or continuous fluid handling system. Also contemplated within each module are in-line sampling ports for in-process analytical testing and/or in-line analytical measurement techniques.

general definition

The following definitions are included to understand the present subject matter and constitute the scope of the appended claims. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

Although various implementations and aspects of the invention have been shown and described herein, it will be apparent to those skilled in the art that these implementations and aspects are provided by way of example only. Now, many modifications, alterations, and substitutions will be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention.

Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, manuals, and treatises, are incorporated by reference in their entirety for any purpose. are expressly incorporated herein.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, eg, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to aid understanding of certain terms frequently used herein and are not intended to limit the scope of the present invention.

Unless specifically stated or otherwise apparent from context, the term “or” as used herein is understood to be inclusive. Unless specifically stated or otherwise apparent from context, the terms "a", "an", and "the" as used herein are to be understood in the singular or plural.

The term "about" when used in reference to a range of numbers, cutoffs, or specific values is used to indicate that the stated value may vary from the recited value by up to 25%. It should be understood by those skilled in the art that, since many of the numerical values used herein have been determined empirically, such determinations can and often will vary between different experiments. The values used herein should not be considered unduly limiting due to these inherent variations. The term "about" means no more than ±25% variation, no more than ±20% variation, no more than 10% variation, no more than ±5% variation, no more than ±1% variation, no more than ±0.5% variation, or Used to include variations of ±0.1% or less. A drug is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. can be understood Unless the context clearly indicates otherwise, all numerical values provided herein are qualified with the term "about".

Ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 60 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, Any number, combination or sub-range of numbers from the group consisting of 46, 47, 48, 49, or 50, as well as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, It is understood to include all intermediate decimal values between the aforementioned integers, such as 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" extending from the endpoints of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 60 include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 50 in the other direction. 30, 50 to 20, and 50 to 10.

As used herein, the term “subject” refers to a living member of the animal kingdom. In embodiments, the subject is a member of a species that includes individuals who are naturally susceptible to the disease. In embodiments, the subject is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs, horses, cats, livestock (e.g., eg, pigs, cattle, donkeys, mules, bison, goats, camels, and sheep). In embodiments, the subject is a human.

As used herein, the term "within the proximity of" or "within the proximity of" means that a distance (i.e., for example, the distance between a magnet or magnetic field and a transport vessel wall or cross-flow channel) is less than about 1 cm; For example, less than about 5 mm.

The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended; Additional unrecited elements or method steps are not excluded. The conjunction phrase "consisting of" excludes any element, step, or ingredient not specified in a claim. The conjunction phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. .

In this specification and claims, phrases such as "at least one of" or "one or more of" may appear after a linked list of elements or features. The term “and/or” can also appear in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which such phrases are used, such phrases mean any of the listed elements or features individually, or any of the recited elements or features. It is intended to mean in combination with other recited elements or features of. For example, "at least one of A and B"; "at least one of A and B"; and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is intended for lists containing three or more items. For example, "at least one of A, B, and C"; “at least one of A, B, and C”; and the phrases "A, B, and/or C" means "A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together", respectively. it is intended to Also, the use of the term "based on" above and in the claims is intended to mean "at least partially based on" such that features or elements not recited are also permitted.

The terms "mechanically smooth" or "substantially smooth" are used interchangeably herein to describe a material's contact surface having a low static coefficient of friction of about 0.01 to about 0.1. .

The terms “hybrid fluidic device” or “hybrid fluidic chip” refer to cross-flow channel dynamics; magnetophoretic dynamics; and either phonophoresis or dielectrophoretic mechanics; are used interchangeably herein to describe a fluid flow device or chip that manipulates magnetic resin beads at high flow rates. Also, the hybrid fluid element may include electrokinetic or capillary flow dynamics. In some aspects of the present disclosure, a hybrid fluidic element or chip includes a cross-flow channel, at least one magnetic field, and at least one piezoelectric component (eg, a piezoelectric crystal). In other aspects of the present disclosure, a hybrid fluidic device or chip includes a cross-flow channel, at least one magnetic field, and at least one dielectrophoretic electrode. A hybrid fluidic device or chip can be a microfluidic, mesofluidic, millifluidic, macrofluidic, or any combination thereof. For example, without intending to be limiting, a hybrid fluidic device can be a microfluidic device that includes a cross-flow channel, at least one magnetic field, and at least one dielectrophoretic electrode.

The terms "tangential flow filtration (TFF) system", "high performance tangential flow filtration (HP-TFF) system", and "cross flow filtration system (CFF)" refer to a sample from a supply vessel in a flow path parallel to the porous membrane face. A solution is supplied so that one fraction passes orthogonally through the membrane (e.g., permeate) and the remaining sample solution is recycled back to the sample supply vessel (e.g., retentate) for microfiltration, ultrafiltration, or diafiltration. , or any combination thereof, are used interchangeably herein to refer to equipment and control systems that allow purification of a sample solution. The membrane may be in the form of a charged or uncharged flat plate or hollow fiber. In addition, tangential flow filtration and cross-flow filtration systems are defined as one-dimensional systems used to purify biomolecules through separation based on 10-fold differences in hydrodynamic size. In contrast, high-performance tangential flow filtration systems are defined as two-dimensional systems that purify biomolecules through separation based on differences in charge characteristics and 10-fold differences in hydrodynamic size.

As used herein, the terms "microfilter", "microfiltrate" or "microfiltration" refer to a TFF process that utilizes a membrane with a pore size greater than 0.1 micron to concentrate resin beads. refers to

As used herein, the term "ultrafilter" or "ultrafiltrate" or "ultrafiltration" refers to a biological product (eg, a protein or fragment thereof (polypeptide), an antibody or fragment thereof). , cytokines, chemokines, enzymes, or growth factors) using membranes with a pore size of less than 0.1 micron.

As used herein, the terms “diafilter,” “diafiltrate,” or “diafiltration” refer to dilution of the residual liquid produced by microfiltration or ultrafiltration with a buffer solution, respectively. Refinement or reultrafiltration refers to a TFF process in which the retentate is further purified or buffer exchange is possible.

As used herein, the term “diavolume” refers to the volume of diafiltration buffer used in a unit TFF run compared to the initial retentate volume.

The terms "free flow electrophoresis" and "isoelectric point based fluid purification" are used interchangeably herein to refer to a continuous flow process that separates biological products based on charge, isoelectric point, electrophoretic mobility, or any combination thereof. used interchangeably. In some aspects of this disclosure, free flow electrophoresis is performed by different electrophoresis including but not limited to isoelectric focusing (IEF-FFE), zone electrophoresis (ZE-FFE), isokinetic electrophoresis, or any combination thereof. has a mode of operation.

As used herein, the term "isoelectric point" or "pI" refers to the pH at which a protein is charge neutral or has no net charge.

A pH gradient as used herein may refer to a "rough pH gradient" or a "fine pH gradient". For example, an approximate pH gradient can have a pH of about 2 to about 10, or about 2 to about 9, or about 2 to about 8, or 2 to about 7, or 2 to about 5, or 2 to about 4, or It refers to a pH range (or gradient) that is from about 2 to about 3. Also, an approximate pH gradient may range from about 5 to about 8 pH. Alternatively, a fine pH gradient refers to a pH gradient (eg, pH change) within 1 pH unit. For example, the pH range is about 7.0 to about 8.0, or about 7.1 to about 8.0, or about 7.2 to about 8.0, or about 7.3 to about 8.0, or about 7.4 to about 8.0, or about 7.4 to about 8.0, or about 7.5 to about 8.0, or about 7.6 to about 8.0, or about 7.7 to about 8.0, or about 7.8 to about 8.0, or about 7.9 to about 8.0. Alternatively, the pH range in the fine pH gradient may range from about 7.0 to about 7.5, or from about 7.1 to about 7.5, or from about 7.3 to about 7.5, or from about 7.4 to about 7.5. In addition, the pH range may be about 7.1 to about 7.6.

As used herein, the term “ampholyte” refers to an amphoteric electrolyte or an electrolyte having both acid and base functional groups. In some aspects of the present specification, an amphoteric electrolyte comprises an amphoteric organic buffer salt or amino acid used to establish a pH gradient in an isoelectric point based fluid purification device. For example, without intending to be limiting, ampholytes include Tris, HEPES, MES, glycine, histidine, arginine, glutamic acid, or any combination thereof.

As used herein, the term "antibody" is used in the broadest sense and is a polypeptide comprising or consisting of antibody domains, to be understood as constant and/or variable domains of heavy and/or light chains of immunoglobulins, with or without linker sequences. or proteins. The term encompasses various antibody constructs including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies such as bispecific antibodies, and antibody fragments. As used herein, the term "monoclonal antibody" refers to the same antibody or antibody fragment produced by a monoclonal cell or cell line that has binding affinity and specificity for a target antigen. Antibody domains may be of natural structure or may be modified by mutagenesis or derivatization. Also, the term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide linked. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. . Similarly, from N- to C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or light chain variable domain, followed by a constant light (CL) domain, also referred to as a light chain constant region. Immunoglobulins of the IgG class consist essentially of two F(ab) domains and an Fc domain linked through an immunoglobulin hinge region. The heavy chain of an immunoglobulin can be of five types derived from any animal (e.g., any commonly used animal, e.g., sheep, rabbit, goat, or mouse), namely IgG, IgM, IgA, and IgE. , and IgD immunoglobulin isotypes, which can be further divided into subtypes such as IgG1, IgG2, or IgG2a. Preferably, the antibody comprises a monoclonal antibody (eg a human monoclonal antibody).

As used herein, the term “valent” indicates the presence of a specific number of binding sites on an antibody molecule. Thus, the terms “bivalent,” “trivalent,” and “multivalent” indicate that an antibody molecule has two binding sites, three binding sites, and multiple binding sites, respectively. The term "monovalent" as used herein with reference to the binding site of an antibody shall refer to a molecule comprising only one binding site for a target antigen. Accordingly, the term “valency” is understood to be the number of binding sites for the same target antigen that specifically bind to the same or different epitopes of the antigen.

The term "monospecific antibody" as used herein refers to the ability of a single antibody to selectively bind to a single individual target with specific and high affinity. Specific and high affinity means that the monospecific antibody preferentially binds to one individual target antigen of interest, while negligible binding to other molecules in the sample solution. Specific binding sites do not typically cross-react with other targets, but specific binding sites can specifically bind to one or more epitopes, isoforms, or variants of a target. By specific binding is meant that the binding is selective in terms of target identity, eg high binding affinity or avidity. Selective binding is generally achieved when the binding constant or binding kinetics to the target antigen is preferably at least 100-fold, more preferably at least 1000-fold greater than the binding constant or binding kinetics to the non-target antigen or molecule. As used herein, the term “high affinity” refers to 10 ^-6 M (micromolar affinity) or less, preferably less than 10 ^-9 M (nanomolar affinity), more preferably less than 10 ^-12 M (picomolar affinity). refers to the binding interaction of an antibody with a target antigen that has an equilibrium dissociation constant (K _D ) of molar affinity. As used herein, "substantially no cross-reactivity" means that the antibody does not recognize or specifically bind to an antigen other than the actual target antigen of the molecule, particularly when compared to the target antigen. For example, the antibody may bind less than about 10% to less than about 5% of an antigen different from its actual target antigen, or about 10%, 9%, 8% 7%, 6%, Less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, most preferably about 0.2% or 0.1 It can bind to an antigen other than the actual target antigen in an amount consisting of less than %. Monospecific antibodies may be of one of four types, specifically human, humanized, chimeric, and murine, and may be derived from IgG, IgM, IgA, IgE, IgD immunoglobulin isotypes and subtypes. In addition, monospecific antibodies may have monovalent, bivalent, trivalent, or multivalent binding sites for the target antigen of interest. Similarly, the term "bispecific antibody" as used herein refers to the ability of a single antibody to be specific for each target and to selectively bind to two different individual targets independently with high affinity. In aspects, specific and high affinity means that the bispecific antibody preferentially binds to two different desired individual target antigens, while negligible binding to other molecules in the sample solution. . Also, the term "trispecific antibody" as used herein refers to the ability of a single antibody to selectively bind to three different individual targets independently with high affinity and specific for each target. In aspects, specific and high affinity means that the trispecific antibody preferentially binds to three different desired individual target antigens, while negligible binding to other molecules in the sample solution. . Multispecific antibodies may also comprise or be formed from antibody fragments.

The term "antibody fragment" as used herein refers to a molecule other than an intact antibody comprising a portion of an intact antibody that binds an antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include monovalent IgGs, linear antibodies, single chain antibody molecules, Fv, scFv, scFv-Fc, F(ab), F(ab)2, scF(ab), F(ab)-scFv fusions. , F(ab)-(scFv)2 fusions, F(ab)-scFv-Fc, cross-F(ab), nanobodies, minibodies, diabodies, scFv-Fc diabodies, and/or affibodies ) are included. Antibody fragments may also have the characteristics of a VH domain, i.e., be capable of assembling together with a VL domain at a functional antigen-binding site, or have the characteristics of a VL domain, i.e., be capable of assembling together with a VH domain at a functional antigen-binding site, such that a full-length antibody is obtained. It includes single chain polypeptides that provide antigen binding properties.

The term “chimeric antibody” refers to an antibody in which part of the heavy and/or light chains are derived from a particular source or species and the remaining heavy and/or light chains are from a different source or species, generally prepared by recombinant DNA techniques. do. A chimeric antibody may comprise a rabbit or murine variable region and a human constant region. Another type of “chimeric antibody” is one in which the constant regions have been modified or altered from those of the original antibody to produce the properties according to the present invention. Such chimeric antibodies are also referred to as "class-switched antibodies". A chimeric antibody is the product of an expressed immunoglobulin gene comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies include conventional recombinant DNA, and gene transfection techniques are well known in the art (Morrison SL, et al., PNAS, 81:6851-6855, (1984)).

As used herein, the term “human antibody” is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cells or derived from a non-human source that utilizes human antibody repertoires or other human antibody encoding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding moieties. As also referred to with chimeric and humanized antibodies, the term "human antibody" as used herein also includes those in which such antibodies have been modified in the constant regions, for example by "class switching".

As used herein, the terms “recombinant antibody” and “recombinant human antibody” refer to an expression host cell, such as, without limitation, a mammalian cell (eg, HEK 293, NSO or CHO cell) or bacterial cell ( Recombinant means, such as antibodies isolated from animals (eg mice) transformed for human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. It is intended to include all human antibodies prepared, expressed, produced, or isolated by These recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibody according to the present invention has undergone in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. As used herein, the term "recombinant" means "produced by genetic engineering" or "the result of genetic engineering", for example, specifically using heterologous sequences integrated into a recombinant vector or recombinant host cell. would mean

The term “humanized antibody” refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions and amino acid residues from human framework regions (FRs) that have been humanized. In certain embodiments, a humanized antibody is one in which all or substantially all of the hypervariable regions (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. , will contain substantially all of at least one, and usually two, variable domains. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. Other forms of humanized antibodies encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to create new properties, such as, for example, Fc receptor binding.

As used herein, the terms "purify", "purified", "purifying" or "purifying" refers to a method of removing impurities from a biological product (eg, nucleic acid, protein, antibody product, etc.) in a heterogeneous mixture. refers to In aspects, impurities include cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, media components, and/or naturally occurring organic molecules with which they are naturally associated. In some aspects, the term "major impurities" or "major impurities" as used herein refers to cells, cell debris, and/or aggregates. In another aspect, the term "small impurities" or "small impurities" as used herein refers to host cell proteins, undesirable proteins and peptides, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants , sugars, metal contaminants, leachates, media components, and/or naturally occurring organic molecules with which they are naturally associated. The purified biological product is at least 60% by weight (dry weight) of the desired product. Preferably, the formulation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the desired product. For example, a purified antibody can contain at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% by weight of the antibody of interest ( w/w). Purity is determined by any suitable standard method, for example column chromatography. A tablet is also defined to a degree of sterility that is safe for administration to a subject, eg, lacking an infectious, toxic, or immunogenic agent. Similarly, “substantially pure” means that a biological product (eg, nucleic acid, protein, antibody product, etc.) is separated from naturally occurring components. In general, biological products (eg, antibodies, proteins, polypeptides, etc.) are impurities (eg, cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable at least 60%, 70%, It is substantially pure when 80%, 90%, 95% or even 99% by weight is removed.

As used herein, the term "isolate", "isolate" or "isolate" refers to the specific selection of a desired biological product (e.g., nucleic acid, protein, antibody product, etc.) from a heterogeneous mixture and Refers to a method of separation from a product. Also, as used herein, an “isolated antibody” refers to an antibody that is substantially free of other antibodies having other antigenic specificities (e.g., an antibody that specifically binds to CD20 and specifically binds to an antigen other than CD20). isolated antibody substantially free). In addition, an isolated antibody may be substantially free of impurities (e.g., cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffers). constituents, surfactants, sugars, metal contaminants, leachates, media constituents, and/or naturally occurring organic molecules with which they are associated naturally).

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids, which in embodiments may be conjugated to moieties that do not consist of amino acids. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. "Fusion" or "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences recombinantly expressed or chemically synthesized as a single moiety.

As used herein, the term "nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides, ribonucleotides, and 2'-modified nucleotides) and polymers thereof in single-stranded, double-stranded, or multi-stranded form, or these refers to the complements of Terms such as "polynucleotide", "oligonucleotide", or "oligo" refer to a linear sequence of nucleotides in their ordinary and customary sense. The term "nucleotide" in its ordinary and conventional sense refers to a single unit of polynucleotide, i.e., a monomer. Nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids (e.g., polynucleotides) contemplated herein include all types of RNA (e.g., mRNA, siRNA, miRNA, and guide RNA) and all types of DNA (e.g., genomic DNA, plasmids, DNA, and minicircle DNA), and any fragments thereof. Also, as used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid oligomer", "oligonucleotide", "nucleic acid sequence", "nucleic acid fragment" and "polynucleotide" are used interchangeably and , deoxyribonucleotides and/or ribonucleotides, and/or analogs, derivatives, or variations thereof, such as polymeric forms of nucleotides covalently linked to each other that may be of various lengths. Different polynucleotides may have different three-dimensional structures and may perform a variety of known and unknown functions. Non-limiting examples of polynucleotides include genomic DNA, genomic, mitochondrial DNA, genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatic DNA), double stranded DNA (dsDNA), messenger RNA (mRNA), transfer RNA, enhancer RNA (eRNA), micro RNA, interfering RNA (RNAi), small interfering RNA (siRNA), ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides Included are nucleotides, plasmids, vectors, aptamers, isolated DNA of sequence, isolated RNA of sequence, nucleic acid probes, and primers. Polynucleotides useful in the methods of the present invention may include natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of such sequences.

As used herein, the term “amino acid” includes both naturally occurring and non-naturally occurring amino acids. For purposes of this invention, naturally occurring amino acids include the 20 naturally occurring L-amino acids. These 20 amino acids can be divided into neutrally charged, positively charged, and negatively charged amino acids. For reference, "neutral" amino acids are listed with respective three-letter and one-letter codes and polarities: Alanine: (Ala, A) non-polar, neutral; Asparagine: (Asn, N) polar, neutral; Cysteine: (Cys, C) non-polar, neutral; Glutamine: (Gln, Q) polar, neutral; Glycine: (Gly, G) non-polar, neutral; Isoleucine: (Ile, I) non-polar, neutral; Leucine: (Leu, L) non-polar, neutral; Methionine: (Met, M) non-polar, neutral; Phenylalanine: (Phe, F) non-polar, neutral; Proline: (Pro, P) non-polar, neutral; Serine: (Ser, S) polar, neutral; Threonine: (Thr, T) polar, neutral; Tryptophan: (Trp, W) non-polar, neutral; Tyrosine: (Tyr, Y) polar, neutral; Valine: (Val, V) non-polar, neutral; and Histidine: (His, H) polar, positively charged (10%) neutral (90%). "Positively charged" amino acids are: Arginine: (Arg, R) polar, positively charged; and Lysine: (Lys, K) polar, positive charge. "Negatively charged" amino acids are: Aspartic acid: (Asp, D) polar, negatively charged; and glutamic acid: (Glu, E) polar, negatively charged. Examples of non-naturally occurring amino acids include D-amino acids (i.e., amino acids having opposite chirality to their naturally occurring form), N-α-methyl amino acids, C-α-methyl amino acids, β-methyl amino acids, and D- or L- β-amino acids, but are not limited thereto. Other non-naturally occurring amino acids include, for example, β-alanine (β-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (γ-Abu), 2 -Aminoisobutyric acid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn), sarcosine, α-aminoisobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3 -diaP), D- or L-phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p-fluorophenylalanine.

The term "continuous" or "semi-continuous" refers to processes in which the production and purification of biological products are carried out with substantially no or no interruptions for extended periods of time, with minor interruptions, or with unintended interruptions. For example, the process of conveying the bioreactor effluent solution to the dynamic filtration module is performed without interruption or with minor interruption. In another example, the process of removing impurities from the heterogeneous mixture by dynamic filtration and passing the filtrate (containing the biological product) to a first module (e.g., an affinity-based purification module) is performed without interruption or with minor interruption. do. Additionally, the process of transferring the solution from the first module to the second module (eg, charge-based purification) is performed without interruption or with minor interruption. In another example, the product stream from dynamic filtration through the first module, the second module, and/or subsequent steps is conducted without interruption or with minor interruption. That is, subsequent unit operations can start processing the product stream before the first unit operation has completed processing the product stream.

The dynamic filtration and/or purification process of the present invention (affinity-based magnetic purification, charge-based magnetic purification, affinity-based purification, charge-based purification, affinity-based fluid purification, charge-based fluid purification, affinity-based TFF purification) module, charge-based TFF purification module, and/or isoelectric point-based purification process), "continuous" means that the processes are physically capable of operating without interruption of fluid flow from a steady-state bioreactor for an extended period of time. and integrated logistically. The processes of the present invention are capable of continuous operation for long periods of time, for example from one day to several months, without disturbing the operation or sequence of the processes. The term continuous when used in connection with the processes of the disclosed invention is also understood to mean that the process is not carried out in a batchwise or truly continuous manner. For example, a process that includes a hold-up volume may be considered continuous if the process can operate without disturbing the fluid flow from the bioreactor discharge line. As used herein, processes are 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. , or for consecutive periods of 3 months, 4 months, 5 months, 6 months or more.

The terms "semi-continuous" and "intermittent" mean that one or more of the processes or elements of an integrated system operates in a discontinuous or batch mode, e.g., a fed-batch mode of operation, while other processes of the integrated system operate. means that the s or elements operate in a continuous manner.

The methods and processes described herein may be continuous, semi-continuous or non-continuous. Minor (and/or unintentional and/or intended) disruptions may include, for example, tears or breaks (eg, in a filter membrane of a dynamic filtration module). For example, tearing or breaking of a filter membrane can include any change that affects the integrity of the filter membrane to perform its function. Any blockage or obstruction in any port, tubing, component, or device throughout the system may be considered a minor disruption. Other minor disruptions considered include an overfilled container/vessel or an underfilled vessel/container. A malfunction of the magnet or magnetic field used during purification may also be considered a minor disruption. A malfunction of the loop conveyor system used during refining may also be considered a minor disruption. A malfunction of the pick-and-place robotic system used during refining may also be considered a minor disruption. A malfunction of the mechanical rotation system used during purification can also be considered a minor disruption. Malfunctions of in-line analytical measuring instruments, such as, for example, sensors or detectors, used during purification may also be considered minor disruptions. A malfunction of a feedback control mechanism used during refinement, such as a PID or closed loop controller for example, may also be considered a minor disruption.

The term “integrated” when used in reference to multiple devices, modules, systems, and/or processes is intended to denote an integrated system in which the devices, modules, systems, and/or processes can operate continuously. It means that it is physically and transportically connected to constitute. In the context of the system of the present invention, which relates to an integrated continuous or semi-continuous system for producing a purified biological product, an integrated system is configured to differ from each other in a manner sufficient to maintain continuous flow between the different components of the system. We will connect the elements directly.

The term “weight percent” or “% (w/w)” refers to the percentage of a component in a solution calculated based on the weight of the component and solvent. For example, a 1% (w/w) solution of a component would represent 1 g of the component dissolved in 100 g of solvent. The term “volume percent” or “% (v/v)” refers to the percentage of a component in a solution calculated based on the volume of the component and solvent. For example, a 1% (v/v) solution of a component would represent 1 ml of the component dissolved in 100 ml of solvent.

Example

The following examples illustrate certain specific embodiments of the invention and are not intended to limit the scope of the invention.

Embodiments herein are further illustrated by the following examples and detailed protocols. However, the examples are only for exemplifying embodiments and should not be construed as limiting the scope of the present invention. The contents of all references and published patents and patent applications cited throughout this application are incorporated herein by reference.

Example 1: Method for Continuous Production and Purification of Monoclonal Antibodies Using a Dynamic Filtration Module, an Affinity-Based Magnetic Purification Module, and a Free Flow Electrophoresis Module

Etanercept is produced continuously in a chemostat bioreactor operating at steady state with a titer of 4 g/L. The heterogeneous mixture comprising etanercept, large impurities, and small impurities is delivered to the dynamic filtration module through a single output head communicating with an input line (perfusion bioreactor discharge line) at a flow rate of 10 mL/min. Large impurities are filtered by dynamic filtration (0.45 μm PES, rolled filter membrane; mechanically flat membrane support structure with parallel slotted openings and temperature control; washing zone; membrane transfer rate 1 mm/s; vacuum gauge pressure -0.9 bar; control 2 vacuum collection vessels with possible T-valves) to produce a filtrate containing etanercept and minor impurities. When the first vacuum collection vessel reaches capacity, flow is diverted to the second vacuum collection vessel and the first vacuum collection vessel equilibrates with atmospheric pressure.

The filtrate is transferred from the first vacuum collection vessel (equalized to atmospheric pressure) through tubing connections and a peristaltic pump to the inlet of the affinity-based magnetic purification module at a flow rate of 10 mL/min. The filtrate was prepared in a thin layer filled with 2 wt % Protein A coated magnetic resin beads (40 μm) suspended in binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH 7) at the home position of the loop conveyor system. It is introduced into the transport container of the wall. Once the transport vessel is full, it is moved to the equilibration zone and is engaged for 30 minutes, during which time the next transport vessel continues to receive a continuous stream of filtrate. Following binding of the antibody to the protein A coated magnetic resin beads, the transport container is moved into a permanent magnetic field region so that the magnetic resin beads can move towards the wall of the transport container. Solutions containing minor impurities are removed by suction and sent to a waste container. Binding/washing buffer (0.025M Tris, 0.15M NaCl; pH 7) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Binding/washing buffer is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Binding/washing buffer is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. A low pH elution buffer (0.1M glycine, pH 2.0) is added and the transport vessel is moved to the equilibration zone to allow elution. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. A low pH elution buffer (0.1M glycine, pH 2.0) is added and the transport vessel is moved to the equilibration zone to allow elution. The transport container moves into the permanent magnetic field region so that the magnetic resin beads can move towards the single wall of the transport container. The solution is removed by suction and sent to a collection vessel. Regeneration buffer (0.25M Tris; pH 8.5) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Binding/washing buffer (0.025M Tris, 0.15M NaCl; pH 7) is added and the transport vessel is moved to the equilibration zone to allow buffer exchange to return the magnetic resin beads to their initial state to complete recycling.

The affinity purified antibody solution is delivered from the collection vessel to the inlet of the isoelectric based fluid purification module at a flow rate of 10 mL/min via a tubing connection and a peristaltic pump. The solution is an amphoteric electrolyte solution designed to achieve a stable linear pH gradient between pH 4 and pH 9 under an applied voltage so that it can operate in isoelectric focusing mode, a degassing and degassing system enabling continuous and long-term operation, Joule heat and an active cooling system to remove and maintain the temperature between 4°C and 37°C, and a liquid circuit breaker to enable in-line process monitoring. The goal of the first device is to separate the remaining host cell proteins (pl 4-7 and 9-10) from the antibodies (pI 7-9) into at least two fractions at the outlet of the device. The outlet(s) containing the antibody fraction become the inlet of a second free flow electrophoresis device in series, while the outlet(s) containing host cell proteins are directed to waste collection. Antibody fractionation consists of a spacer solution and two separate amphoteric electrolyte solutions designed to operate in a highly resolving isokinetic mode of electrophoresis, a degassing and degassing system allowing continuous and long-term operation, Joule heat removal and temperature control. A second free flow electrophoretic device is introduced which includes an active cooling system to maintain between 4°C and 37°C, and a liquid circuit breaker to enable in-line process monitoring. This goal of the first device is to separate residual host cell antibody (pI 7-9) from etanercept (pI 7.9) into at least two fractions at the outlet of the device. The outlet(s) containing purified etanercept are collected, while the outlet(s) containing antibody impurities are sent to waste collection.

The purified and isolated etanercept solution is transferred from the collection vessel to a high-performance tangential flow filtration (HP-TFF) vessel via tubing connections and a peristaltic pump at a flow rate of 5 mL/min so that HP-TFF can be performed semi-continuously in fed-batch mode. there is. HP-TFF is performed to perform buffer exchange, rituximab is further purified (diafiltered with a diavolume of 10) and then concentrated to allow subsequent vial filling of the retentate containing purified etanercept.

This process is performed continuously for 3 months after reaching steady state cell culture growth conditions.

Example 2: Continuous Production and Purification Method of Monoclonal Antibodies Using Dynamic Filtration Module, Affinity-Based Purification Module, and Free Flow Electrophoresis Module

Etanercept is produced continuously in a chemostat bioreactor operating at steady state with a titer of 4 g/L. The heterogeneous mixture comprising etanercept, large impurities, and small impurities is delivered to the dynamic filtration module through a single output head communicating with an input line (perfusion bioreactor discharge line) at a flow rate of 10 mL/min. Large impurities are filtered by dynamic filtration (0.45 μm PES, rolled filter membrane; mechanically flat membrane support structure with parallel slotted openings and temperature control; washing zone; membrane transfer rate 1 mm/s; vacuum gauge pressure -0.9 bar; control 2 vacuum collection vessels with possible T-valves) to produce a filtrate containing etanercept and minor impurities. When the first vacuum collection vessel reaches capacity, flow is diverted to the second vacuum collection vessel and the first vacuum collection vessel equilibrates with atmospheric pressure.

The filtrate is transferred from the first vacuum collection vessel (equalized to atmospheric pressure) through tubing connections and a peristaltic pump to the inlet of the affinity-based purification module at a flow rate of 10 mL/min. Via a gasket lid system, the filtrate was passed through a cassette filled with 40 mL of a high-density slurry of Protein A coated resin beads (90 μm) suspended in binding/wash buffer (0.025 M Tris, 0.15 M NaCl, pH 7) at the filling position of a mechanical rotation system. It is introduced into the container in the russell. Once the vessel is filled, the vessel is moved to the equilibrium position and allowed to engage for 30 minutes, during which time the next vessel continues to receive a continuous stream of filtrate. Following binding of the protein A coated resin beads to the antibody, the vessel is moved to a washing position to remove the solution containing small impurities by pressure driven flow through the underlying porous membrane and direct it to waste collection. Resuspend the resin beads by adding binding/washing buffer (0.025M Tris, 0.15M NaCl; pH 7) and allow to wash for 5 minutes. The wash solution is removed by pressure driven flow through the underlying porous membrane and sent to waste collection. This washing process is repeated 4 times to effectively clean the resin beads. After washing the protein A coated resin beads, the container is moved to the elution position to elute the captured etanercept by pressure driven flow through the underlying porous membrane, which is sent to a collection container. Resuspend the resin beads by adding low pH elution buffer (0.1 M glycine; pH 2.0) and equilibrate for 5 minutes to allow debinding and elution of etanercept. The eluate is removed by pressure driven flow through the underlying porous membrane and sent to a collection vessel. By repeating this elution process 4 times, etanercept is effectively eluted from the resin beads. After eluting the etanercept from the resin beads, the container can be moved to a regeneration position to recycle the resin beads. The resin beads can be resuspended by adding regeneration buffer (0.25M Tris; pH 8.5) and allowed to equilibrate for 5 minutes to regenerate the resin beads. By repeating this regeneration process twice, the resin beads are effectively regenerated. Binding/washing buffer (0.025M Tris, 0.15M NaCl; pH 7) is added and equilibrated for 5 minutes to allow buffer exchange to return the resin beads to their initial state and complete recycling. This buffer exchange is repeated twice and represents the final aspect of the regeneration and recycling process.

The affinity purified antibody solution is delivered from the collection vessel to the inlet of the isoelectric based fluid purification module at a flow rate of 10 mL/min via a tubing connection and a peristaltic pump. The solution is an amphoteric electrolyte solution designed to achieve a stable linear pH gradient between pH 4 and pH 9 under an applied voltage so that it can operate in isoelectric focusing mode, a degassing and degassing system enabling continuous and long-term operation, Joule heat and an active cooling system to remove and maintain the temperature between 4°C and 37°C, and a liquid circuit breaker to enable in-line process monitoring. The goal of the first device is to separate the remaining host cell proteins (pl 4-7 and 9-10) from the antibodies (pI 7-9) into at least two fractions at the outlet of the device. The outlet(s) containing the antibody fraction become the inlet of a second free flow electrophoresis device in series, while the outlet(s) containing host cell proteins are directed to waste collection. Antibody fractionation consists of a spacer solution and two separate amphoteric electrolyte solutions designed to operate in a highly resolving isokinetic mode of electrophoresis, a degassing and degassing system allowing continuous and long-term operation, Joule heat removal and temperature control. A second free flow electrophoretic device is introduced which includes an active cooling system to maintain between 4°C and 37°C, and a liquid circuit breaker to enable in-line process monitoring. This goal of the first device is to separate residual host cell antibody (pI 7-9) from etanercept (pI 7.9) into at least two fractions at the outlet of the device. The outlet(s) containing purified etanercept are collected, while the outlet(s) containing antibody impurities are sent to waste collection.

The purified and isolated etanercept solution is transferred from the collection vessel to a high-performance tangential flow filtration (HP-TFF) vessel via tubing connections and a peristaltic pump at a flow rate of 5 mL/min so that HP-TFF can be performed semi-continuously in fed-batch mode. there is. HP-TFF is performed to perform buffer exchange, rituximab is further purified (diafiltered with a diavolume of 10) and then concentrated to allow subsequent vial filling of the retentate containing purified etanercept.

This process is performed continuously for 3 months after reaching steady state cell culture growth conditions.

Example 3: Method for Continuous Production and Purification of Monoclonal Antibodies Using a Dynamic Filtration Module, an Affinity-Based Magnetic Purification Module, and a Charge-Based Magnetic Purification Module

Etanercept is produced continuously in a chemostat bioreactor operating at steady state with a titer of 4 g/L. The heterogeneous mixture containing etanercept, large impurities, and small impurities is passed to the dynamic filtration module through a single output head communicating with an input line (chemostat bioreactor discharge line) at a flow rate of 5 mL/min. Large impurities are filtered by dynamic filtration (0.45 μm PES, rolled filter membrane; mechanically flat membrane support structure with parallel slotted apertures and temperature control; washing zone; membrane transfer rate 2 mm/sec; vacuum 6 Torr; controllable T-valve 2 vacuum collection vessels) to produce a filtrate containing etanercept and minor impurities. When the first vacuum collection vessel reaches capacity, flow is diverted to the second vacuum collection vessel and the first vacuum collection vessel equilibrates with atmospheric pressure.

The filtrate is transferred from the first vacuum collection vessel (equalized to atmospheric pressure) through tubing connections and a peristaltic pump to the inlet of the affinity-based magnetic purification module at a flow rate of 5 mL/min. The filtrate consists of 2 wt % Protein A coated magnetic resin beads (40 μm) suspended in binding/wash buffer (0.025 M Tris, 0.15 M NaCl, 0.05% Tween-20; pH 7) at the home position of the loop conveyor system. It is introduced into a filled, thin-walled transport vessel. Once the transport vessel is full, it is moved to the equilibration zone and is engaged for 30 minutes, during which time the next transport vessel continues to receive a continuous stream of filtrate. Following binding of the antibody to the protein A coated magnetic resin beads, the transport container is moved into a permanent magnetic field region so that the magnetic resin beads can move towards the walls of the transport container. The solution is removed by suction and sent to a waste container. Binding/washing buffer (0.025M Tris, 0.15M NaCl, 0.05% Tween-20; pH 7) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Binding/washing buffer is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Binding/washing buffer is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. A low pH elution buffer (0.1 M glycine, 0.05% Tween-20, pH 2.0) is added and the transport vessel is moved to the equilibration zone to allow elution. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. A low pH elution buffer (0.1 M glycine, 0.05% Tween-20, pH 2.0) is added and the transport vessel is moved to the equilibration zone to allow elution. The transport container moves into the permanent magnetic field region so that the magnetic resin beads can move towards the single wall of the transport container. The solution is removed by suction and sent to a collection vessel. Regeneration buffer (0.25M Tris, 0.05% Tween-20; pH 8.5) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. The magnetic resin beads can be recycled by adding a regeneration buffer and moving the transport vessel to the equilibration zone.

The affinity-purified antibody solution is adjusted to pH 7 and transferred from the collection vessel to the inlet of the positive charge-based magnetic purification module via a tubing connection and a peristaltic pump at a flow rate of 5 mL/min. The solution was prepared in a thin-walled transport container filled with 2 wt % cationic magnetic resin beads (40 μm) suspended in association/wash buffer (0.025 M Tris, 0.05% Tween-20, pH 7) at the home position of the loop conveyor system. introduced into Once the transport vessel is filled, it is moved to an equilibration zone to allow charge or electrostatic association for 30 minutes, during which time the next transport vessel continues to receive the continuous flow of affinity purified antibody solution. Following binding of the cationic magnetic resin beads to the antibody, the transport container is moved to a permanent magnetic field region where the magnetic resin beads are mixed between two separate opposing magnetic fields that switch between on and off states, then the transport container to move towards the single wall of The solution is removed by suction and sent to a waste container. Association/wash buffer (0.025M Tris, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Association/wash buffer (0.025M Tris, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Association/wash buffer (0.025M Tris, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Dissociation buffer (0.1M Tris, 0.1M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Dissociation buffer (0.025M Tris, 0.15M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Dissociation buffer (0.025M Tris, 0.2M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Dissociation buffer (0.025M Tris, 0.25M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Regeneration buffer (0.025M Tris, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. The magnetic resin beads can be recycled by adding regeneration buffer (0.025M Tris, 0.05% Tween-20, pH 7) and moving the transport vessel to the equilibration zone.

The positively charged purified antibody solution is buffer exchanged with 0.05 M phosphate, pH 7 by tangential flow filtration and then transferred from the collection vessel to the inlet of the negatively charged based magnetic purification module via a tubing connection and a peristaltic pump at a flow rate of 5 mL/min. do. The solution is a thin-walled transport vessel filled with 2 wt % anionic magnetic resin beads (40 μm) suspended in association/wash buffer (0.05 M phosphate, 0.05% Tween-20, pH 7) at the home position of the loop conveyor system. introduced into Once the transport vessel is filled, the transport vessel is moved to an equilibrium zone to allow charge or electrostatic association for 30 minutes, during which time the next transport vessel continues to receive a continuous flow of positively charged purified antibody solution. Following association of the antibodies with the anionic magnetic resin beads, the transport container is moved into a permanent magnetic field region so that the magnetic resin beads can move towards the wall of the transport container. The solution is removed by suction and sent to a waste container. Association/wash buffer (0.05 M Phosphate, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Association/wash buffer (0.05 M Phosphate, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Association/wash buffer (0.05 M Phosphate, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. Dissociation buffer (0.05 M Phosphate, 0.1 M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Dissociation buffer (0.05 M Phosphate, 0.15 M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Dissociation buffer (0.05 M Phosphate, 0.2 M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Dissociation buffer (0.05 M Phosphate, 0.25 M NaCl, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow dissociation. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a collection vessel. Regeneration buffer (0.05 M Phosphate, 0.05% Tween-20, pH 7) is added and the transport vessel is moved to the equilibration zone to allow for washing. The transport vessel moves into the permanent magnetic field region, causing the magnetic resin beads to mix between two separate opposing magnetic fields that switch between on and off states, and then move towards the single wall of the transport vessel. The solution is removed by suction and sent to a waste container. The magnetic resin beads can be recycled by adding regeneration buffer (0.05 M Phosphate, 0.05% Tween-20, pH 7) and moving the transport vessel to the equilibration zone.

Negatively charged purified and isolated etanercept solution is transferred from the collection vessel to a high performance tangential flow filtration (HP-TFF) vessel via tubing connections and a peristaltic pump at a flow rate of 5 mL/min where HP-TFF is performed semi-continuously in fed-batch mode It can be. HP-TFF is performed to perform buffer exchange, rituximab is further purified (diafiltered with a diavolume of 10) and then concentrated to allow subsequent vial filling of the retentate containing purified etanercept.

This process is performed continuously for 3 months after reaching steady state cell culture growth conditions.

Example 4: Dynamic Filtration Module for Purifying POLYBEADS in Heterogeneous Mixtures

An exemplary dynamic filtration module described herein includes different cell and cell debris mimicking sizes suspended in a 0.5 g/L solution of human polyclonal IgG (hlgG) in 1X PBS at an input flow rate of 10 mL/min from a single slot die output head. (0.5μm, 0.75μm, 1μm, 2μm, 3μm, and 10μm diameter at 7.3x10 ⁷ , 1.1x10 ⁸ , 1.1x10 ⁸ , 1.1x10 ⁸ , 3.4x10 ⁷ , and 1.0x10 ⁶ particles/mL, respectively) provided for continuous dynamic filtration that successfully purified a model target antibody, human polyclonal IgG (hlgG), from a heterogeneous mixture comprising Purification of the PolyBeads resulted in a filtrate containing purified hIgG. Protein recovery at a flow rate of 10 mL/min was comparable to that of a standard 5 min centrifugation process at 10,000 xg (FIGS. 11c and 11d).

The design and material selection of the membrane support structure was critical to enable continuous membrane transport at a rate of 0.5 mm/s under sufficient negative pressure (gauge pressure -0.9 bar) in the wet state, and thus, for all membrane contact surfaces, the wet The selection of a material with a low static friction coefficient (e.g., mechanically flat PTFE) was very important (FIG. 8).

Example 5: Dynamic filtration module for purification of cells from heterogeneous mixtures

An exemplary dynamic filtration module described herein comprises a heterogeneous suspension comprising a suspension of murine myeloma cells (2.0x10 ⁶ cells/mL) in RPMI medium spiked with hlgG at an input flow rate of 2 mL/min from a single slot die output head. The mixture was provided for continuous dynamic filtration which successfully purified the model target antibody, human polyclonal IgG (hlgG), to a final concentration of 1 g/L. Purification of cells and cellular debris produced a filtrate containing purified hIgG. Protein recovery at a flow rate of 2 mL/min was comparable to that of a standard 5 min centrifugation process at 10,000 xg (FIGS. 13A-13C).

The design and material selection of the membrane support structure was critical to enable continuous membrane transport at a rate of 0.5 mm/s under sufficient negative pressure (gauge pressure -0.9 bar) in the wet state, and thus, for all membrane contact surfaces, the wet The selection of a material with a low static friction coefficient (e.g., mechanically flat PTFE) was very important (FIG. 8).

Example 6: Recovery of proteins with different physicochemical properties by dynamic filtration module

Dynamic filtration through an exemplary dynamic filtration module equipped with a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec was performed using different concentrations of BSA (0.5-10 g/L, MW 66,000 Da, pl 4.5-5) solutions, lysozyme solution (5 g/L, MW 14,000 Da, pl 11), and hIgG solution (0.5 g/L, MW 150,000, pl 6-8) at an input flow rate of 10 mL/min, and vacuum gauge pressure of -0.9 bar. protein recovery was assessed, which was determined by spectrophotometric analysis of the filtrate generated by the BCA assay (n=3 for each solution). Protein recovery was similar for all proteins and was observed to exceed 96% (FIG. 12A).

Example 7: Effect of filter membrane material on dynamic filtration module performance

Dynamic filtration through an exemplary dynamic filtration module with different low protein binding filter membrane materials (PES, hydrophilic PVDF) and pore sizes (0.45 μm, 0.22 μm) with a transport rate of 0.5 mm/sec, hIgG solution ( 0.5 g/L) at an input flow rate of 10 mL/min and a vacuum gauge pressure of -0.9 bar to evaluate protein recovery, which was determined by spectrophotometric analysis of the filtrate generated by the BCA assay (n=3 for each solution). determined by analysis. Protein recoveries were similar for each filter membrane material and pore size and were observed to exceed 96% (FIG. 12B).

Example 8: Impact of different membrane support structure shapes and materials on dynamic filtration module performance

Dynamic through an exemplary dynamic filtration module equipped with a mechanically flat PTFE membrane support structure with different opening shapes (5 parallel slots, porous hydrophilic PE insert), and a 0.45 μm PES filter membrane with a transport speed of 0.5 mm/sec. Filtration was performed with an input flow rate of 10 mL/min and a vacuum gauge pressure of -0.9 bar for hIgG solutions (0.5 g/L) to assess protein recovery, which was obtained from the filtrate produced by the BCA assay (n for each solution). = 3) was determined by spectrophotometric analysis. Protein recovery was similar for each membrane support construct and was observed to exceed 96% (FIG. 12C).

Example 9: Continuous and long-term dynamic filtration module performance

Continuous dynamic filtration through an exemplary dynamic filtration module equipped with a mechanically flat PTFE membrane support structure and a 0.45 μm PES filter membrane with a transport rate of 0.5 mm/sec, for lysozyme solution (0.5 g/L) at 5 mL/min or Longitudinal protein recovery was assessed over 25 minutes by an input flow rate of 10 mL/min and vacuum gauge pressure of -0.9 bar, which was determined by spectrophotometric analysis of the filtrate produced by the BCA assay. Protein recovery was similar for each membrane support construct and was observed to exceed 96% (FIG. 12D).

Example 10: Affinity Based Magnetic Purification of Polyclonal Human IgG in Mixtures

The exemplary affinity-based magnetic purification module described herein was used to purify hIgG from a mixture comprising 2 g/L hIgG (affinity target) and 1 g/L lysozyme (small impurity). 4 ml of a mixture containing 8 mg hIgG and 4 mg lysozyme in binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH 7) was mixed with precipitated high-affinity protein A/G magnetic agarose (precipitated resin at >40 mg hIgG/mL). of dynamic binding capacity) was added at 10 mL/min to a thin-walled container filled with 500 μL and allowed to equilibrate for 30 minutes with gentle mixing to allow binding. After 30 minutes of binding equilibrium, a permanent Nd magnet is placed in a thin-walled vessel (e.g., a pick-and-place It was manually placed in close proximity to the robot system). After aspiration, the vessel was filled with 4 mL of binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min and allowed to equilibrate for 5 minutes with gentle mixing for washing. After 5 minutes of cleaning, a permanent Nd magnet is manually placed in close proximity to the thin-walled vessel (e.g., mimicking a pick-and-place robotic system) to attract the magnetophilic beads to the vessel wall and collect the cleaning solution by suction. placed as The washing step was repeated for a total of 3 washes. After aspirating the wash fractions, the vessel was filled with 4 mL of low pH elution buffer (0.1M glycine; pH 2) at 10 mL/min and allowed to equilibrate for 10 minutes with gentle mixing for elution. After 10 minutes of elution, a permanent Nd magnet is manually placed in close proximity to the thin-walled vessel (e.g., mimicking a pick-and-place robotic system) to attract the magnetophilic beads to the vessel wall and collect the eluate fraction by aspiration. placed as The elution step was repeated for a total of 3 elutions. After collecting the three eluate fractions, the vessel was filled with 4 mL of low pH elution buffer (0.1 M glycine; pH 2) at 10 mL/min and allowed to equilibrate for 5 min with gentle mixing to completely remove any remaining bound hIgG. Removal initiated regeneration of the magnetic affinity beads. After 5 minutes of retentate elution, a permanent Nd magnet is placed in a thin-walled vessel (e.g., mimicking a pick-and-place robotic system) so that magnetophilic beads can be attracted to the vessel wall and the first regeneration solution can be collected by aspiration. placed manually in close proximity to After collecting the first regeneration solution, the vessel was filled with 4 mL of regeneration buffer (0.25M Tris; pH 8.5) at 10 mL/min and equilibrated for 5 minutes with gentle mixing to neutralize the pH of the magnetic affinity beads, Magnetic affinity beads were regenerated by removing all remaining hlgG and small impurities. After 5 minutes of regeneration, a permanent Nd magnet is placed in close proximity to the thin-walled vessel (e.g., mimicking a pick-and-place robotic system) so that the magnetophilic beads can be attracted to the vessel wall and a second regeneration solution can be collected by aspiration. placed manually. After collecting the second regeneration solution, the vessel was filled with 4 mL of the second regeneration buffer (0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min and allowed to equilibrate for 5 minutes with gentle mixing to obtain magnetic affinity beads. were buffer exchanged, and the magnetic affinity beads were returned to their initial state. After 5 minutes of regeneration, a permanent Nd magnet is placed in close proximity to the thin-walled vessel (e.g., mimicking a pick-and-place robotic system) to attract the magnetophilic beads to the vessel wall and collect the buffer exchange solution by aspiration. Placed manually. The buffer exchange step was repeated a total of two times to reuse the magnetic affinity beads. Fractions collected for three successive process cycles and magnetic affinity bead recycling were analyzed spectrophotometrically by BCA and found to be robust and reproducible (FIG. 20A). Fractions collected for three consecutive process cycles and magnetic affinity bead recycling were further characterized by SDS-PAGE to confirm reproducibility and showed the ability to purify hIgG (FIG. 20B).

Example 11: Affinity-based purification of polyclonal human IgG from mixtures

The exemplary affinity-based purification module described herein was used to purify hIgG from a mixture comprising 2 g/L hIgG (affinity target) and 1 g/L lysozyme (small impurity). 4 ml of a mixture containing 8 mg of hIgG and 4 mg of lysozyme in binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH 7) was added at 10 mL/min to a vessel containing a base glass frit via a lid system, and precipitated high 500 μL of pyrogenic protein A agarose (90 μm, dynamic binding capacity of the precipitated resin with hIgG/mL greater than 35 mg) was charged and allowed to equilibrate for 30 minutes with gentle mixing to allow binding. After 30 minutes of binding equilibration, compressed air was introduced into the vessel at about 1 psi to collect the solution containing unbound hIgG and small impurities by a pressure driven flow. After collection, the vessel was filled with 4 mL of binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min and allowed to equilibrate for 5 minutes with gentle mixing for washing. After 5 minutes of washing, compressed air was introduced at about 1 psi into the vessel so that the washing solution could be collected by a pressure driven flow. The washing step was repeated for a total of 3 washes. After collecting the three wash fractions, the vessel was filled with 4 mL of low pH elution buffer (0.1M glycine; pH 2) at 10 mL/min and allowed to equilibrate for 10 minutes with gentle mixing for elution. After 10 minutes of elution, the eluate fraction could be collected by pressure driven flow by introducing compressed air at about 1 psi into the vessel. The elution step was repeated for a total of 3 elutions. After collecting the three eluate fractions, the vessel was filled with 4 mL of low pH elution buffer (0.1 M glycine; pH 2) at 10 mL/min and allowed to equilibrate for 5 min with gentle mixing to completely remove any remaining bound hIgG. Regeneration of the affinity resin beads was initiated by removal. After 5 minutes of retentate elution, the first regeneration solution could be collected by introducing compressed air at about 1 psi into the vessel. After collecting the first regeneration solution, the vessel was filled with 4 mL of regeneration buffer (0.25 M Tris; pH 8.5) at 10 mL/min and equilibrated for 5 minutes with gentle mixing to neutralize the pH of the affinity resin beads; , the affinity resin beads were regenerated by removing all remaining hlgG and small impurities. After 5 minutes of regeneration, a second regeneration solution could be collected by introducing compressed air at about 1 psi into the vessel. After collecting the second regeneration solution, the vessel was filled with 4 mL of the second regeneration buffer (0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min and allowed to equilibrate for 5 minutes with gentle mixing to release the affinity resin beads. The buffer was exchanged, and the affinity resin beads were returned to their initial state. After 5 minutes of regeneration, the first regeneration solution could be collected by introducing compressed air at about 1 psi into the vessel. The buffer exchange step was repeated a total of 2 times to allow the affinity resin beads to be reused. Fractions collected for three successive process cycles and magnetic affinity bead recycling were analyzed spectrophotometrically by BCA and found to be robust and reproducible (FIG. 27A). Fractions collected for three consecutive process cycles and magnetic affinity bead recycling were further characterized by SDS-PAGE to confirm reproducibility and showed the ability to purify hIgG (FIG. 27B).

Example 12: Separation of Small Molecules by Isoelectric Focusing Free Flow Electrophoresis at High Flow Rate Using Isoelectric Point-based Fluid Purification Module

A mixture of rhodamine 6G (0.25 mg/mL) and fluorescein (0.25 mg/mL) was prepared under an applied voltage to operate in the positive channel (H ₂ SO ₄ ), negative channel (NaOH), and isoelectric focusing mode. A main separation channel with 5 inlets and 5 outlets through which the ampholyte solution flows, designed to achieve a stable linear pH gradient between pH 2 and pH 12, a degassing and degassing system allowing continuous and long-term operation, and a bottom plate A central inlet (inlet) of an exemplary free flow electrophoresis device including an active cooling system (cooled circulating ethylene glycol/water containing heat chuck) to remove Joule heat and maintain a temperature between 4° C. and 37° C. 3) was introduced. When no voltage was applied, the mixture exited the device at the central outlet (outlet 3) following a laminar flow (FIGS. 40a and 40b). When 1000 V was applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 10 mL/min, a linear pH gradient was established, and rhodamine 6G and fluorescein migrated to the cathode and anode, respectively, which correspond to the theoretical electrophoretic migration also matched the predictions (FIGS. 40c and 40d). Spectrophotometric analysis of the fractions collected from outlet 2 and outlet 4 showed the presence of purified rhodamine 6G and purified fluorescein, respectively (FIG. 40e).

Example 13: Separation of small molecules by isokinetic electrophoresis at high flow rate using an isoelectric point-based fluid purification module

A mixture of rhodamine 6G (0.25 mg/mL) and fluorescein (0.25 mg/mL) was prepared as a basic amphoteric to allow operation in the positive channel (H ₂ SO ₄ ), negative channel (NaOH), and isoelectric focusing mode. Main separation channels with 5 inlets and 5 outlets through which the electrolyte solution (inlets 1 and 2), the spacer solution (inlets 3) and the acidic ampholyte solution (inlets 4 and 5) flow, enabling continuous and long-term operation An exemplary free flow including a bubble removal and degassing system and an active cooling system (heat chuck with chilled circulating ethylene glycol/water) to remove joule heat through the lower plate and maintain the temperature between 4°C and 37°C. It was introduced into the central inlet (inlet 3) of the electrophoresis device. When no voltage was applied, the mixture exited the device at the central outlet (outlet 3) following a laminar flow. When 250 V was applied to the main separation channel with an ampholyte at a sample input flow rate of 5 mL/min, rhodamine 6G and fluorescein migrated to the cathode and anode, respectively, resulting in highly concentrated, highly concentrated, and highly resolved bands. formed (Figs. 44a and 44b).

Example 14: Separation of Basic and Acidic Small Molecules by Isoelectric Focusing Free Flow Electrophoresis at High Flow Rate Using Isoelectric Point-based Fluid Purification Module

A mixture of basic fuchsine (0.05mg/mL) and fluorescein (0.25mg/mL) or crystal violet (0.05mg/mL) and fluorescein (0.25mg/mL) was added to the anode channel (H ₂ SO ₄ ), Main separation with a cathodic channel (NaOH) and five inlets and five outlets through which an ampholytic solution designed to achieve a stable linear pH gradient between pH 2 and pH 12 under applied voltage to allow operation in isoelectric focusing mode Channels, degassing and degassing system to enable continuous and long-term operation, and active cooling system to remove joule heat through the bottom plate and maintain temperature between 4°C and 37°C (with chilled circulating ethylene glycol/water was introduced into the central inlet (inlet 3) of an exemplary free flow electrophoresis device containing a thermal chuck). When no voltage was applied, the mixture exited the device at the central outlet (outlet 3) following a laminar flow. When 500 V was applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 5 mL/min, a linear pH gradient was established, and basic fuchsine and fluorescein migrated to the cathode and anode, respectively, which correspond to the theoretical electrophoretic mobility Consistent with predictions (FIG. 42A). Similarly, when 500 V was applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 5 mL/min, a linear pH gradient was established, and crystal violet and fluorescein migrated to the cathode and anode, respectively, which correspond to the theoretical electrical The migration mobility was also consistent with the prediction (FIG. 42B).

Example 15: Effect of Increasing Electric Field on Separation of Basic and Acidic Small Molecules by Isoelectric Focusing Free Flow Electrophoresis at High Flow Rate Using Isoelectric Point-based Fluid Purification Module

A mixture of basic fuchsin (0.005 mg/mL) and fluorescein (0.25 mg/mL) is an amphoteric acid designed to achieve a stable linear pH gradient between pH 2 and pH 12 under an applied voltage allowing it to operate in isoelectric focusing mode. Main separation channels with 5 inlets and 10 outlets with electrolyte solution, anode and cathode channels with the same amphoteric electrolyte as the separation channels, degassing and degassing system enabling continuous and long-term operation, liquid circuit breakers , and an active cooling system (heat chuck with cooled circulating ethylene glycol/water) to remove Joule heat through the lower plate and maintain the temperature between 4°C and 37°C. introduced into the inlet (inlet 3). When no voltage was applied, the mixture exited the device at the central outlet (outlets 4 and 5) following a laminar flow (FIG. 43A). When voltage was applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 5 mL/min, a linear pH gradient was established, and basic fuchsin and fluorescein migrated to the cathode and anode, respectively, which corresponded to the theoretical electrophoretic mobility Consistent with predictions (FIGS. 43B to 43D). As the electric field strength was increased by increasing the applied voltage from 600 V (FIG. 43B) to 900 V (FIG. 43C) and 1100 V (FIG. 43D), the separation of the two molecules was observed to increase proportionally to the length of the main separation channel.

Example 16: Separation of Acidic and Basic Proteins by Isoelectric Focusing Free Flow Electrophoresis at High Flow Rate Using Isoelectric Point-based Fluid Purification Module

A mixture of BSA (0.5 mg/mL, pI 4-5) and lysozyme (0.25 mg/mL, pI 11) can be operated in positive channel (H ₂ SO ₄ ), negative channel (NaOH), and isoelectric focusing mode. Main separation channel with 5 inlets and 5 outlets through which the ampholyte solution flows, designed to achieve a stable linear pH gradient between pH 2 and pH 12 under an applied voltage to ensure continuous, long-term operation, degassing and degassing system, and an active cooling system (heat chuck with chilled circulating ethylene glycol/water) to remove Joule heat through the lower plate and maintain the temperature between 4°C and 37°C. It was introduced into the central inlet (inlet 3). When no voltage was applied, the mixture exited the device at the central outlet (Outlet 3) following a laminar flow (FIGS. 45A and 45B). When 850 V was applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 10 mL/min, a linear pH gradient was established, and lysozyme and BSA migrated to the cathode and anode, respectively (FIGS. 45c and 45d), which is consistent with the theoretical electrical The migration mobility was also consistent with the prediction (FIG. 45E).

Example 17: Separation of human polyclonal IgG by high-flow isoelectric focusing free flow electrophoresis using an isoelectric point-based fluid purification module

A mixture of hIgG (0.5 mg/mL, pI 6-8) and lysozyme (0.25 mg/mL, pI 11) can be operated in positive channel (H ₂ SO ₄ ), negative channel (NaOH), and isoelectric focusing mode. Main separation channel with 5 inlets and 5 outlets through which the ampholyte solution flows, designed to achieve a stable linear pH gradient between pH 2 and pH 12 under an applied voltage to ensure continuous, long-term operation, degassing and degassing system, and an active cooling system (heat chuck with chilled circulating ethylene glycol/water) to remove Joule heat through the lower plate and maintain the temperature between 4°C and 37°C. It was introduced into the central inlet (inlet 3). When no voltage was applied, the mixture exited the device at the central outlet (outlet 3) following a laminar flow (FIGS. 46a and 46c). When 1000 V was applied to the main separation channel with an amphoteric electrolyte with a sample input flow rate of 5 mL/min, a linear pH gradient was established and migration of lysozyme to the cathode was observed (FIGS. 46a and 46c), which was consistent with the theoretical electrophoretic migration. also matched the predictions (FIG. 46B). Increasing the applied voltage to 1500 V increased the migration of lysozyme to the cathode. This increase in applied voltage also shifted hIgG to the cathode and anode (FIGS. 46A and 46C), consistent with theoretical electrophoretic mobility predictions for the polyclonal antibody-specific Pls range (FIG. 46B).

Other embodiments

Although the present invention has been described with detailed description, the foregoing description is illustrative and not limiting of the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patents and scientific literature cited herein establish knowledge available to those skilled in the art. All references cited herein, eg, US patents, US patent application publications, PCT patent applications designating the US, published foreign patents and patent applications, are incorporated herein by reference in their entirety. Genbank and NCBI submissions indicated by accession numbers cited herein are hereby incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Although the invention has been shown and described with reference to particularly preferred embodiments, those skilled in the art will appreciate that various changes in form and detail may be made without departing from the scope of the invention as encompassed by the appended claims.

Claims (30)

A method for purifying a biological product, the method comprising:
receiving a heterogeneous mixture comprising a biological product through an input line;
removing impurities from the heterogeneous mixture by filtration in a dynamic filtration module by supplying the biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module; generating a filtrate comprising biological products, wherein the dynamic filtration module extends between a feed reel and a collection reel together with at least one support member having a substantially planar contact surface. a dynamic filtration device having a filter membrane that is substantially in communication with a target area of the filter membrane configured to receive the heterogeneous mixture from at least one output head, and a vacuum collection system positioned between the supply reel and the collection reel. comprising a membrane support member having a flat contact surface;
passing the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains the biological product, and the first module comprises an affinity-based purification device. wherein the first module is between at least one first inlet and at least one first outlet via a mechanical rotating system comprising a container carousel containing at least one individual container containing a suspension of beads. having at least one first inlet and at least one first outlet configured to allow flow of a fluid of;
passing the fraction comprising the biological product from at least one outlet of the first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module; - the second module comprises at least one free-flow electrophoresis device, wherein the second module has at least one second inlet and at least one second outlet, the second inlet and configured to allow continuous fluid flow between the second outlet; and
Recovering the biological product; method comprising a. The method of claim 1 , wherein the affinity-based purification device further comprises a lid system and a collection vessel system in fluid communication with the at least one individual vessel. 3. The method of claim 2, wherein the lid system includes at least one lid with gasket, at least two buffer inlets, a filling inlet, a gas inlet, and a venting valve. 3. The method of claim 2, wherein the lid system is movable along the z-axis, the container carousel is rotationally movable in a plane transverse to the z-axis, and the collection container is movable along the z-axis. The method of claim 1 , wherein the container carousel has at least one location for binding the biological product, at least one location for washing to remove unbound products, and at least one location for eluting and collecting the biological product. a location, and at least one regeneration location enabling recycling of the beads. The method of claim 1, wherein the surface of the beads are linked to protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer configured to selectively bind to the biological product. , method. The method of claim 1 , wherein the initial concentration of the beads ranges from about 0.01% to about 25% by weight. The method of claim 1 , wherein the diameter of the beads ranges from about 0.2 μm to about 200 μm. The method of claim 1 , wherein the beads remain mobile during the process to maintain increased surface area available for bonding. 2. The free flow electrophoretic device of claim 1, wherein the free flow electrophoresis device includes electrode channels including an anode electrode channel and a cathode electrode channel in liquid contact with a main separation channel through a wall gap, The apparatus includes at least one electrode channel de-bubbler comprising at least one gas permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a primary bubble-free separation channel, and at least one liquid and further having a liquid circuit breaker. 2. The method of claim 1 , wherein the method maintains an approximately constant flow rate in the dynamic filtration module, the first module, and the second module, wherein the flow rate ranges from about 0.1 mL/min to about 50 mL/min. , method. The method of claim 1 , wherein the method of purifying the biological product is performed at a temperature ranging from about 4° C. to about 37° C. The method of claim 1 , wherein the method further comprises at least two dynamic filtration modules, wherein each dynamic filtration module has a filter membrane comprising the same or different pore size. The method of claim 1 , wherein the method comprises at least two free flow electrophoresis modules configured to operate in an isoelectric focusing mode, a zone electrophoresis mode, an isotachophoresis mode, or a combination thereof. Further comprising them, the method. The method of claim 1 , wherein the method further comprises at least two dynamic filtration modules, at least two affinity based purification modules, or at least two free flow electrophoresis modules operated in parallel. A dynamic filtration device for removing impurities from a biological product in a heterogeneous mixture, the device comprising:
a filter membrane extending between the supply reel and the collection reel, the filter membrane having a target area configured to receive the heterogeneous mixture from at least one output head configured to distribute the heterogeneous mixture to the target area;
a membrane support structure having a substantially planar contact surface for structurally supporting a portion of the filter membrane positioned between the supply reel and the collection reel to create the target area;
at least one support member having a substantially planar contact surface for stabilizing transport of the filter membrane across the membrane support structure;
a system configured to control a transport rate of the filter membrane;
a vacuum system comprising at least one vacuum line in communication with the membrane support structure and configured to apply a negative pressure gauge pressure to the dynamic filter membrane, wherein the negative pressure allows the filtrate containing the biological product to be collected; , a dynamic filtration device. 17. The apparatus of claim 16, wherein the apparatus further comprises a wash buffer line. 17. The method of claim 16, wherein the filter membrane is polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene ( PTFE), hydrophilic PTFE, or any combination thereof. 17. The device of claim 16, wherein the filter membrane comprises a pore size in the range of about 0.1 μm to about 1 μm. 17. The apparatus of claim 16, wherein the membrane support structure comprises a series of parallel slots. 17. The device of claim 16, wherein the substantially flat contact surface has a static coefficient of friction of about 0.01 to about 0.1. 17. The apparatus of claim 16, wherein the gauge pressure ranges from about -0.05 bar to about -0.98 bar. A free flow electrophoretic device for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product, the device comprising:
at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet;
at least one fluid channel configured to create an electric field gradient created between the two parallel plates and perpendicular to the direction of fluid flow;
electrode channels comprising an anode electrode channel and a cathode electrode channel, wherein the electrode channels are configured to be connected to the main separation channel by liquid contact through a wall gap located between the electrode channels and the main separation channel;
at least one electrode channel bubble eliminator comprising at least one gas permeable and hydrophobic membrane or porous material configured to remove electrolytic bubbles near the point of creation by a vacuum system to create a primary bubble free separation channel;
at least one liquid circuit breaker configured to disconnect the liquid connected to the voltage prior to interacting with the at least one sensor or detector;
active cooling system; and
A free flow electrophoresis device comprising: at least one collection vessel. 24. The method of claim 23, wherein the upper portion of the electrode channels are sealed with at least one gas permeable and hydrophobic membrane in communication with a vacuum system for removing air bubbles, the electrode channels being open at the bottom of the channels, and the wall and configured to enable liquid contact of the electrode solution with the primary separation channel solution through a gap. 24. The device of claim 23, wherein the wall gap is between about 0.01 mm and about 0.25 mm. 24. The apparatus of claim 23, wherein the liquid circuit breaker includes a pressurized vessel configured to generate droplets that maintain the flow rate and disconnect the circuit from the solution coupled to a voltage. 24. The apparatus of claim 23, wherein the at least one sensor is an in-line sensor. 24. The method of claim 23, wherein the device is connected in series to enable staged purification, and is operated in isoelectric focusing mode, bandelectrophoresis mode, isokinetic mode, or a combination thereof. The device further comprising free flow electrophoresis devices. Use of the device according to claim 16 for the purification of biological products from heterogeneous mixtures. Use of the device according to claim 23 for the purification of a biological product from a mixture.

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